Materials and methods for treatment of hereditary haemochromatosis

ABSTRACT

Materials and methods for treating a patient with hereditary hemochromatosis (HHC), both ex vivo and in vivo, and materials and methods for modulating the expression, function, or activity of a haemochromatosis (HFE) gene in a cell by genome editing.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Patent Application Serial No. PCT/IB2017/000317, filedMar. 16, 2017, which claims priority to U.S. Provisional ApplicationSer. No. 62/323,890, filed Apr. 18, 2016, and U.S. ProvisionalApplication Ser. No. 62/309,136, filed Mar. 16, 2016, the contents ofeach of which are incorporated by reference herein in their entirety.

FIELD

The present application provides materials and methods for treating apatient with hereditary haemochromatosis (HHC), both ex vivo and invivo. In addition, the present application provides materials andmethods for modulating the expression, function, and/or activity of ahaemochromatosis (HFE) gene in a cell by genome editing.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form(filename: 160073PCT sequence listing_ST25: 13,042,115 bytes—ASCII textfile; created Mar. 13, 2017), which is incorporated herein by referencein its entirety and forms part of the disclosure.

BACKGROUND

HHC is characterized by excessive storage of iron in the liver, skin,pancreas, heart, joints, and testes. It remains the most common,identified, genetic disorder in Caucasians. Although its geographicdistribution is worldwide, it is most common in populations of northernEuropean origin, particularly Nordic or Celtic ancestry, in which itoccurs with a prevalence of approximately 1 per 220-250 individuals. Thepathophysiologic predisposition to increased, inappropriate absorptionof dietary iron may lead to the development of life-threateningcomplications such as cirrhosis, hepatocellular carcinoma (HCC),diabetes, and heart disease.

In 1996, HFE, a gene involved in HHC, was located on Chromosome 6(6p21.3 region) and found to contain 7 exons spanning 12 kb.

It was discovered that the HFE gene has multiple allelic variants. Oneknown mutation is a G-to-A missense mutation leading to the substitutionof tyrosine for cysteine at amino acid position 282 of the proteinproduct (C282Y). C282Y homozygotes account for 80-85% of typicalpatients with HHC. Allele frequencies of HFE C282Y in ethnically diversewestern European white populations are 5-14% and in North Americannon-Hispanic whites are 6-7%. C282Y exists as a polymorphism only inWestern European white and derivative populations, although C282Y mayhave arisen independently in non-whites outside Europe. There are twoother regularly identified mutations of the HFE gene, one in whichaspartate is substituted for histidine at amino acid position 63 (H63D),and the other in which cysteine is substituted for serine at amino acidposition 65 (S65C). These mutations are generally not associated withiron loading unless seen with C282Y as a compound heterozygote,C282Y/H63D or C282Y/S65C.

Over the last 10 years, mutations of other genes coding for ironregulatory proteins have been implicated in inherited iron overloadsyndromes (e.g., hepcidin, hemojuvelin, transferrin receptor 2, andferroportin). These mutations of other genes coding for iron regulatoryproteins are thought to account for most of the non-HFE forms of HHC.

The largest predicted primary translation product of the HFE gene is 348amino acids, which gives rise to a mature protein of approximately 321amino acids after cleavage of the signal sequence. The HFE protein issimilar to HLA Class I molecules at the level of the primary structureand tertiary structure. The mature protein is expressed on the cellsurface as a heterodimer with beta-2-microglobulin, and this interactionis necessary for normal presentation on the cell surface. The C282Ypathogenic variant destroys a key cysteine residue that is required fordisulfide bonding with beta-2-microglobulin. As a result, the HFEprotein does not mature properly and becomes trapped in the endoplasmicreticulum and Golgi apparatus, leading to decreased cell-surfaceexpression.

The diagnosis of HHC in individuals is typically based on findingelevated transferrin-iron saturation 45% or higher and serum ferritinconcentration above the upper limit of normal (i.e., >300 ng/mL in menand >200 ng/mL in women) and two pathogenic variants on confirmatory HFEmolecular genetic testing. Although serum ferritin concentration mayincrease progressively over time in untreated individuals with HHC, itis not specific for HHC, and therefore cannot be used alone foridentification of individuals with HHC.

For patients diagnosed with HHC, treatment by phlebotomy (removal ofblood) is an available option to help maintain serum ferritinconcentration at 50 ng/mL.

An alternative treatment for patients diagnosed with HHC includes genomeengineering. Genome engineering refers to the strategies and techniquesfor the targeted, specific modification of the genetic information(genome) of living organisms. Genome engineering is a very active fieldof research because of the wide range of possible applications,particularly in the areas of human health; the correction of a genecarrying a harmful mutation, for example, or to explore the function ofa gene. Early technologies developed to insert a transgene into a livingcell were often limited by the random nature of the insertion of the newsequence into the genome. Random insertions into the genome may resultin disrupting normal regulation of neighboring genes leading to severeunwanted effects. Furthermore, random integration technologies offerlittle reproducibility, as there is no guarantee that the sequence wouldbe inserted at the same place in two different cells. Recent genomeengineering strategies, such as ZFNs, TALENs, HEs and MegaTALs, enable aspecific area of the DNA to be modified, thereby increasing theprecision of the correction or insertion compared to early technologies.These newer platforms offer a much larger degree of reproducibility, butstill have their limitations.

Despite efforts from researchers and medical professionals worldwide whohave been trying to address HHC, there still remains a critical need fordeveloping safe and effective treatments for HHC.

Currently, phlebotomy treatment is the only available treatment foraddressing HHC, and it only aims to manage symptoms, not treat thecause.

SUMMARY

The present disclosure presents an approach to address the genetic basisof HHC. By using genome engineering tools to create permanent changes tothe genome that can address the HFE gene and restore HFE proteinactivity with as few as a single treatment, the resulting therapy mayameliorate the effects of or completely eliminate HHC

Provided herein are cellular, ex vivo and in vivo methods for creatingpermanent changes to the genome by deleting, inserting, correcting, ormodulating the expression or function of one or more mutations within ornear an HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene, which can be used to treat HHC. Also provided hereinare components, kits, and compositions for performing such methods. Alsoprovided are cells produced by such methods.

Provided herein is a method for editing an HFE gene in a human cell bygenome editing, the method comprising: introducing into the human cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the HFE gene or other DNA sequences that encode regulatoryelements of the HFE gene that results in a permanent deletion,insertion, correction, or modulation of expression or function of one ormore mutations within or near or affecting the expression or function ofthe HFE gene and results in restoration of HFE protein activity. Thehuman cell can be a liver cell, skin cell, pancreatic cell, heart cell,joint cell, or cell from the testes.

Also provided herein is a method for inserting a haemochromatosis (HFE)gene in a human cell by genome editing, the method comprising:introducing into the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near a safe harbor locus thatresults in a permanent insertion of the HFE gene, and results inrestoration of HFE protein activity.

Also provided herein is an ex vivo method for treating a patient (e.g.,a human) with HHC, the method comprising: creating a patient specificinduced pluripotent stem cell (iPSC); editing within or near an HFE geneor other DNA sequences that encode regulatory elements of the HFE geneof the iPSC, or editing within or near a safe harbor locus of the iPSC;differentiating the genome-edited iPSC into a hepatocyte; and implantingthe hepatocyte into the patient.

The step of creating a patient specific induced pluripotent stem cell(iPSC) can comprise: isolating a somatic cell from the patient; andintroducing a set of pluripotency-associated genes into the somatic cellto induce the somatic cell to become a pluripotent stem cell. Thesomatic cell can be a fibroblast. The set of pluripotency-associatedgenes can be one or more of the genes selected from the group consistingof OCT4, SOX2, KLF4, Lin28, NANOG and cMYC.

The step of editing within or near an HFE gene or other DNA sequencesthat encode regulatory elements of the HFE gene of the iPSC or editingwithin or near a safe harbor locus of the iPSC can comprise: introducinginto the iPSC one or more deoxyribonucleic acid (DNA) endonucleases toeffect one or more single-strand breaks (SSBs) or double-strand breaks(DSBs) within or near the HFE gene or other DNA sequences that encoderegulatory elements of the HFE gene that results in a permanentdeletion, insertion, correction, or modulation of expression or functionof one or more mutations within or near or affecting the expression orfunction of the HFE gene, or within or near a safe harbor locus, thatresults in restoration of HFE protein activity.

The safe harbor locus can be selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

The step of differentiating the genome-edited iPSC into a hepatocyte cancomprise: contacting the genome-edited iPSC with one or more of activin,B27 supplement, FGF4, HGF, BMP2, BMP4, Oncostatin M, or Dexametason.

The step of implanting the hepatocyte into the patient can comprise:implanting the hepatocyte into the patient by transplantation, localinjection, systemic infusion, or combinations thereof.

Also provided herein is an ex vivo method for treating a patient (e.g.,a human) with HHC, the method comprising: performing a biopsy of thepatient's liver; isolating a liver specific progenitor cell or primaryhepatocyte from the patient's liver; editing within or near an HFE geneor other DNA sequences that encode regulatory elements of the HFE geneof the liver specific progenitor cell or primary hepatocyte or editingwithin or near a safe harbor locus of the liver specific progenitor cellor primary hepatocyte; and implanting the genome-edited liver specificprogenitor cell or primary hepatocyte into the patient.

The step of isolating a liver specific progenitor cell or primaryhepatocyte from the patient's liver can comprise perfusion of freshliver tissues with digestion enzymes, cell differencial centrifugation,cell culturing, or combinations thereof.

The step of editing within or near an HFE gene or other DNA sequencesthat encode regulatory elements of the HFE gene of the liver specificprogenitor cell or primary hepatocyte or editing within or near a safeharbor locus of the liver specific progenitor cell or primary hepatocytecan comprise: introducing into the liver specific progenitor cell orprimary hepatocyte one or more deoxyribonucleic acid (DNA) endonucleasesto effect one or more single-strand breaks (SSBs) or double-strandbreaks (DSBs) within or near the HFE gene or other DNA sequences thatencode regulatory elements of the HFE gene or within or near a safeharbor locus that results in a permanent deletion, insertion,correction, or modulation of expression or function of one or moremutations within or near or affecting the expression or function of theHFE gene, or within or near a safe harbor locus, that results inrestoration of HFE protein activity.

The safe harbor locus can be selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

The step of implanting the genome-edited liver specific progenitor cellor primary hepatocyte into the patient can comprise: implanting thegenome-edited liver specific progenitor cell or primary hepatocyte intothe patient by transplantation, local injection, systemic infusion, orcombinations thereof.

Also provided herein is an ex vivo method for treating a patient (e.g.,a human) with HHC, the method comprising: isolating a mesenchymal stemcell from the patient; editing within or near an HFE gene or other DNAsequences that encode regulatory elements of the HFE gene of themesenchymal stem cell, or editing within or near a safe harbor locus ofthe mesenchymal stem cell; differentiating the genome-edited mesenchymalstem cell into a hepatocyte; and implanting the hepatocyte into thepatient.

The mesenchymal stem cell can be isolated from the patient's bone marrowby performing a biopsy of the patient's bone marrow or the mesenchymalstem cell can be isolated from peripheral blood. The step of isolating amesenchymal stem cell from the patient can comprise aspiration of bonemarrow and isolation of mesenchymal cells using density gradientcentrifugation media.

The step of editing within or near the HFE gene or other DNA sequencesthat encode regulatory elements of the HFE gene of the mesenchymal stemcell can comprise introducing into the mesenchymal stem cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene that results in a permanent deletion, insertion,correction, or modulation of expression or function of one or moremutations within or near or affecting the expression or function of theHFE gene or within or near a safe harbor locus, that results inrestoration of HFE protein activity.

The safe harbor locus can be selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

The step of differentiating the genome-edited mesenchymal stem cell intoa hepatocyte can comprise contacting the genome-edited mesenchymal stemcell with one or more of insulin, transferrin, FGF4, HGF, or bile acids.

The step of implanting the hepatocyte into the patient can compriseimplanting the hepatocyte into the patient by transplantation, localinjection, systemic infusion, or combinations thereof.

Also provided herein is an in vivo method for treating a patient (e.g.,a human) with HHC, the method comprising the step of editing an HFE genein a cell of the patient or other DNA sequences that encode regulatoryelements of the HFE gene, or editing within or near a safe harbor locusin a cell of the patient. The cell can be a liver cell, skin cell,pancreatic cell, heart cell, joint cell, or cell from the testes.

The step of editing an HFE gene in a cell of the patient can compriseintroducing into the cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene that resultsin a permanent deletion, insertion, correction, or modulation ofexpression or function of one or more mutations within or near oraffecting the expression or function of the HFE gene or within or near asafe harbor locus that results in restoration of HFE protein activity.

The one or more DNA endonucleases can be a Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of thenaturally occurring molecule thereof, codon-optimized thereof, ormodified versions thereof, and combinations thereof.

The method can comprise introducing into the cell one or morepolynucleotides encoding the one or more DNA endonucleases. The methodcan comprise introducing into the cell one or more ribonucleic acids(RNAs) encoding the one or more DNA endonucleases. The one or morepolynucleotides or one or more RNAs can be one or more modifiedpolynucleotides or one or more modified RNAs. The DNA endonuclease canbe one or more proteins or polypeptides.

The method can further comprise introducing into the cell one or moreguide ribonucleic acids (gRNAs). The one or more gRNAs can besingle-molecule guide RNA (sgRNAs). The one or more gRNAs or one or moresgRNAs can be one or more modified gRNAs, one or more modified sgRNAs,or combinations thereof. The one or more DNA endonucleases can bepre-complexed with one or more gRNAs, one or more sgRNAs, orcombinations thereof.

The method can further comprise introducing into the cell apolynucleotide donor template comprising at least a portion of thewild-type HFE gene, DNA sequences that encode wild-type regulatoryelements of the HFE gene, and/or cDNA. The at least a portion of thewild-type HFE gene or cDNA can be exon 1, exon 2, exon 3, exon 4, exon5, exon 6, exon 7, intronic regions, fragments of combinations thereof,or the entire HFE gene or cDNA. The donor template can be either singleor double stranded. The donor template can have homologous arms to the6p21.3 region.

The method can further comprise introducing into the cell one guideribonucleic acid (gRNA) and a polynucleotide donor template comprisingat least a portion of the wild-type HFE gene. The method can furthercomprise introducing into the cell one guide ribonucleic acid (gRNA) anda polynucleotide donor template comprising at least a portion of a codonoptimized or modified HFE gene. The one or more DNA endonucleases can beone or more Cas9 or Cpf1 endonucleases that effect one single-strandbreak (SSB) or double-strand break (DSB) at a locus within or near theHFE gene (or codon optimized or modified HFE gene) or other DNAsequences that encode regulatory elements of the HFE gene, or within ornear a safe harbor locus that facilitates insertion of a new sequencefrom the polynucleotide donor template into the chromosomal DNA at thelocus or safe harbor locus that results in a permanent insertion orcorrection of a part of the chromosomal DNA of the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene proximal tothe locus, or safe harbor locus. The gRNA can comprise a spacer sequencethat is complementary to a segment of the locus. Proximal can meannucleotides both upstream and downstream of the locus or safe harborlocus.

The method can further comprise introducing into the cell one or moreguide ribonucleic acid (gRNAs) and a polynucleotide donor templatecomprising at least a portion of the wild-type HFE gene. The one or moreDNA endonucleases can be one or more Cas9 or Cpf1 endonucleases thateffect or create at least two (e.g., a pair) single-strand breaks (SSBs)and/or double-strand breaks (DSBs), the first at a 5′ locus and thesecond at a 3′ locus, within or near the HFE gene or other DNA sequencesthat encode regulatory elements of the HFE gene or within or near a safeharbor locus that facilitates insertion of a new sequence from thepolynucleotide donor template into the chromosomal DNA between the 5′locus and the 3′ locus that results in a permanent insertion orcorrection of the chromosomal DNA between the 5′ locus and the 3′ locuswithin or near the HFE gene or other DNA sequences that encoderegulatory elements of the HFE gene or within or near a safe harborlocus. The first guide RNA can comprise a spacer sequence that iscomplementary to a segment of the 5′ locus and the second guide RNA cancomprise a spacer sequence that is complementary to a segment of the 3′locus.

The one or more gRNAs can be one or more single-molecule guide RNA(sgRNAs). The one or more gRNAs or one or more sgRNAs can be one or moremodified gRNAs or one or more modified sgRNAs. The one or more DNAendonucleases can be pre-complexed with one or more gRNAs or one or moresgRNAs.

The at least a portion of the wild-type HFE gene or cDNA can be exon 1,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, intronic regions,fragments, or combinations thereof, or the entire HFE gene or cDNA.

The donor template can be either a single or double strandedpolynucleotide. The donor template can have homologous arms to the6p21.3 region.

The gRNA or sgRNA can be directed to one or more of the pathologicalvariants: C282Y, H63D, or S65C or combinations thereof.

The SSB or DSB can be in the first, second, third, fourth, fifth, sixth,seventh exon, or combinations thereof or introns of the HFE gene.

The insertion or correction can be by homology directed repair (HDR).

The method can further comprise introducing into the cell two guideribonucleic acids (gRNAs). The one or more DNA endonucleases can be oneor more Cas9 or Cpf1 endonucleases that effect or create two or more(e.g., a pair) double-strand breaks (DSBs), the first at a 5′ DSB locusand the second at a 3′ DSB locus, within or near the HFE gene or otherDNA sequences that encode regulatory elements of the HFE gene, or withinor near a safe harbor locus that causes a deletion of the chromosomalDNA between the 5′ DSB locus and the 3′ DSB locus that results in apermanent deletion of the chromosomal DNA between the 5′ DSB locus andthe 3′ DSB locus within or near the HFE gene or other DNA sequences thatencode regulatory elements of the HFE gene or safe harbor locus. Thefirst guide RNA can comprise a spacer sequence that is complementary toa segment of the 5′ DSB locus and the second guide RNA comprises aspacer sequence that is complementary to a segment of the 3′ DSB locus.

The two gRNAs can be two single-molecule guide RNA (sgRNAs). The twogRNAs or two sgRNAs can be two modified gRNAs or two modified sgRNAs.The one or more DNA endonucleases can be pre-complexed with one or twogRNAs or one or two sgRNAs.

The 5′ DSB and/or 3′ DSB can be in or near the first exon, first intron,second exon, second intron, third exon, third intron, fourth exon,fourth intron, fifth exon, fifth intron, sixth exon, sixth intron,seventh exon, seventh intron, or combinations thereof, of the HFE gene.

The deletion can be a deletion of 1 kb or less.

The Cas9 or Cpf1 mRNA, gRNA, and donor template can be formulated intoseparate lipid nanoparticles or co-formulated into a lipid nanoparticle.

The Cas9 or Cpf1 mRNA can be formulated into a lipid nanoparticle, andthe gRNA and donor template can be delivered to the cell by anadeno-associated virus (AAV) vector.

The Cas9 or Cpf1 mRNA can be formulated into a lipid nanoparticle, andthe gRNA can be delivered to the cell by electroporation and donortemplate can be delivered to the cell by an adeno-associated virus (AAV)vector.

The HFE gene can be located on Chromosome 6: 26087458-26095569 (GenomeReference Consortium—GRCh38/hg38).

The restoration of HFE protein activity can be compared to a control(e.g., wild-type or normal HFE protein activity).

Also provided herein is one or more guide ribonucleic acids (gRNAs) forediting an HFE gene in a cell from a patient with HHC. The one or moregRNAs and/or sgRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 1-64,980 ofthe Sequence Listing. The one or more gRNAs can be one or moresingle-molecule guide RNAs (sgRNAs). The one or more gRNAs or one ormore sgRNAs can be one or more modified gRNAs or one or more modifiedsgRNAs. The cell can be a liver cell, skin cell, pancreatic cell, heartcell, joint cell, or cell from the testes.

It is understood that the inventions described in this specification arenot limited to the examples summarized in this Summary. Various otheraspects are described and exemplified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods for treatment of HHC disclosedand described in this specification can be better understood byreference to the accompanying figures, in which:

FIG. 1A is a depiction of the type II CRISPR/Cas system;

FIG. 1B is a depiction of the type II CRISPR/Cas system;

FIG. 2A describes the cutting efficiencies of gRNAs selected via anin-vitro transcribed (IVT) gRNA screen;

FIG. 2B describes the cutting efficiencies of gRNAs selected via anin-vitro transcribed (IVT) gRNA screen;

FIG. 2C describes the cutting efficiencies of gRNAs selected via anin-vitro transcribed (IVT) gRNA screen;

FIG. 2D describes the cutting efficiencies of gRNAs selected via anin-vitro transcribed (IVT) gRNA screen;

FIG. 3A describes the cutting efficiency of S. pyogenes gRNAs in HEK293Tcells targeting the HFE gene;

FIG. 3B describes the cutting efficiency of S. pyogenes gRNAs in HEK293Tcells targeting the HFE gene; and

FIG. 3C describes the cutting efficiency of S. pyogenes gRNAs in HEK293Tcells targeting the HFE gene.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-4,455 are 20 bp spacer sequences for targeting an HFE genewith an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 4,456-4,979 are 20 bp spacer sequences for targeting an HFEgene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 4,980-5,139 are 20 bp spacer sequences for targeting an HFEgene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 5,140-5,192 are 20 bp spacer sequences for targeting an HFEgene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 5,193-5,617 are 20 bp spacer sequences for targeting an HFEgene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 5,618-10,121 are 20-24 bp spacer sequences for targeting anHFE gene with an Acidominococcus, a Lachnospiraceae, and a FranciscellaNovicida Cpf1 endonuclease.

SEQ ID NOs: 10,122-12,153 are 20 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 12,154-12,324 are 20 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 12,325-12,342 are 20 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with an S. thermophilus Cas9endonuclease.

SEQ ID NOs: 12,343-12,351 are 20 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 12,352-12,426 are 20 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with an N. meningitides Cas9endonuclease.

SEQ ID NOs: 12,427-13,602 are 22 bp spacer sequences for targeting exons1-2 of an AAVS1 (PPP1R12C) gene with an Acidominococcus,Lachnospiraceae, and Francisella novicida Cpf1 endonuclease.

SEQ ID NOs: 13,603-13,770 are 20 bp spacer sequences for targeting exons1-2 of an Alb gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 13,771-13,798 are 20 bp spacer sequences for targeting exons1-2 of an Alb gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 13,799-13,816 are 20 bp spacer sequences for targeting exons1-2 of an Alb gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 13,817-13,821 are 20 bp spacer sequences for targeting exons1-2 of an Alb gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 13,822-13,845 are 20 bp spacer sequences for targeting exons1-2 of an Alb gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 13,846-14,224 are 22 bp spacer sequences for targeting exons1-2 of an Alb gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 14,225-14,569 are 20 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 14,570-14,605 are 20 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 14,606-14,628 are 20 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 14,629-14,641 are 20 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 14,642-14,704 are 20 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 14,705-15,552 are 22 bp spacer sequences for targeting exons1-2 of an Angpt13 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 15,553-15,955 are 20 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 15,956-15,980 are 20 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 15,981-15,983 are 20 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 15,984-15,985 are 20 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 15,986-15,997 are 20 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 15,998-16,229 are 22 bp spacer sequences for targeting exons1-2 of an ApoC3 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 16,230-17,997 are 20 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 17,998-18,203 are 20 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 18,204-18,227 are 20 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 18,228-18,239 are 20 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 18,240-18,322 are 20 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 18,323-19,762 are 22 bp spacer sequences for targeting exons1-2 of an ASGR2 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 19,763-19,965 are 20 bp spacer sequences for targeting exons1-2 of a CCR5 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 19,966-19,997 are 20 bp spacer sequences for targeting exons1-2 of a CCR5 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 19,998-20,011 are 20 bp spacer sequences for targeting exons1-2 of a CCR5 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 20,012-20,013 are 20 bp spacer sequences for targeting exons1-2 of a CCR5 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 20,014-20,041 are 20 bp spacer sequences for targeting exons1-2 of a CCR5 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 20,042-20,341 are 22 bp spacer sequences for targeting exons1-2 of a CCR5 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 20,342-21,807 are 20 bp spacer sequences for targeting exons1-2 of an F9 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 21,808-21,970 are 20 bp spacer sequences for targeting exons1-2 of an F9 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 21,971-22,031 are 20 bp spacer sequences for targeting exons1-2 of an F9 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 22,032-22,056 are 20 bp spacer sequences for targeting exons1-2 of an F9 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 22,057-22,209 are 20 bp spacer sequences for targeting exons1-2 of an F9 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 22,210-24,350 are 22 bp spacer sequences for targeting exons1-2 of an F9 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 24,351-25,366 are gRNA 20 bp spacer sequences for targetingexons 1-2 of the G6PC gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 25,367-25,483 are gRNA 20 bp spacer sequences for targetingexons 1-2 of the G6PC gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 25,484-25,507 are gRNA 20 bp spacer sequences for targetingexons 1-2 of the G6PC gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 25,508-25,516 are gRNA 20 bp spacer sequences for targetingexons 1-2 of the G6PC gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 25,517-25,606 are gRNA 20 bp spacer sequences for targetingexons 1-2 of the G6PC gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 25,607-26,701 are gRNA 22 bp spacer sequences for targetingexons 1-2 of the G6PC gene with an Acidominococcus, Lachnospiraceae, and

Francisella novicida Cpf1 endonuclease.

SEQ ID NOs: 26,702-32,194 are 20 bp spacer sequences for targeting exons1-2 of an Gys2 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 32,195-32,870 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Gys2 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 32,871-33,148 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Gys2 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 33,149-33,262 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Gys2 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 33,263-33,942 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Gys2 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 33,943-42,374 are gRNA 22 bp spacer sequences for targetingexons 1-2 of an Gys2 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 42,375-44,067 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an HGD gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 44,068-44,281 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an HGD gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 44,282-44,364 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an HGD gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 44,365-44,383 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an HGD gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 44,384-44,584 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an HGD gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 44,585-46,909 are gRNA 22 bp spacer sequences for targetingexons 1-2 of an HGD gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 46,910-50,704 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Lp(a) gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 50,705-51,114 are gRNA 20 bp spacer sequences for exons 1-2of targeting the Lp(a) gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 51,115-51,250 are gRNA 20 bp spacer sequences for targetingthe exons 1-2 of Lp(a) gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 51,251-51,285 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Lp(a) gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 51,286-51,653 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an Lp(a) gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 51,654-56,274 are gRNA 22 bp spacer sequences for targetingexons 1-2 of an Lp(a) gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 56,275-58,294 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a PCSK9 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 58,295-58,481 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a PCSK9 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 58,482-58,517 are gRNA 20 bp spacer sequences, for targetingexons 1-2 of a PCSK9 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 58,518-58,531 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a PCSK9 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 58,532-58,671 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a PCSK9 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 58,672-60,465 are gRNA 22 bp spacer sequences for targetingexons 1-2 of a PCSK9 gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 60,466-61,603 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 61,604-61,696 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 61,697-61,708 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 61,709-61,711 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 61,712-61,762 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 61,763-62,566 are gRNA 22 bp spacer sequences for targetingexons 1-2 of a Serpina1 gene with an Acidominococcus, Lachnospiraceae,and Francisella novicida Cpf1 endonuclease.

SEQ ID NOs: 62,567-63,398 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TF gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 63,399-63,484 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TF gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 63,485-63,496 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TF gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 63,497-63,503 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TF gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 63,504-63,547 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TF gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 63,548-64,183 are gRNA 22 bp spacer sequences targetingexons 1-2 of a TF gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 64,184-64,483 are gRNA 20 bp spacer sequences for targetingexons 1-2 of an TTR gene with an S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 64,484-64,524 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TTR gene with an S. aureus Cas9 endonuclease.

SEQ ID NOs: 64,525-64,541 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TTR gene with an S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 64,542-64,543 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TTR gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 64,544-64,578 are gRNA 20 bp spacer sequences for targetingexons 1-2 of a TTR gene with an N. meningitides Cas9 endonuclease.

SEQ ID NOs: 64,579-64,980 are gRNA 22 bp spacer sequences for targetingexons 1-2 of a TTR gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

DETAILED DESCRIPTION

Hereditary Haemochromatosis (HHC)

HFE, a gene involved in HHC, is located on Chromosome 6 (6p21.3 region)and contains 7 exons spanning 12 kb.

It was discovered that the HFE gene has multiple allelic variants. Oneknown mutation is a G-to-A missense mutation leading to the substitutionof tyrosine for cysteine at amino acid position 282 of the proteinproduct (C282Y). C282Y homozygotes account for 80%-85% of typicalpatients with HHC. Allele frequencies of HFE C282Y in ethnically diversewestern European white populations are 5-14% and in North Americannon-Hispanic whites are 6-7%. C282Y exists as a polymorphism only inWestern European white and derivative populations, although C282Y mayhave arisen independently in non-whites outside Europe. There are twoother regularly identified mutations of the HFE gene, one in whichaspartate is substituted for histidine at amino acid position 63 (H63D),and the other in which cysteine is substituted for serine at amino acidposition 65 (S65C). These mutations are generally not associated withiron loading unless seen with C282Y as a compound heterozygote,C282Y/H63D or C282Y/S65C.

Mutations of other genes coding for iron regulatory proteins have beenimplicated in inherited iron overload syndromes (e.g., hepcidin,hemojuvelin, transferrin receptor 2, and ferroportin). These othermutated genes are thought to account for most of the non-HFE forms ofHHC.

Therapeutic Approach

As the known forms of HHC are monogenic disorders with recessiveinheritance, it is likely that correcting one of the mutant alleles percell will be sufficient for correction and restoration or partialrestoration of HFE function. The correction of one allele can coincidewith one copy that remains with the original mutation, or a copy thatwas cleaved and repaired by non-homologous end joining (NHEJ) andtherefore was not properly corrected. Bi-allelic correction can alsooccur. Various editing strategies that can be employed for specificmutations are discussed below.

Correction of one or possibly both of the mutant alleles provides animportant improvement over existing or potential therapies, such asintroduction of HFE expression cassettes through lentivirus delivery andintegration. Gene editing has the advantage of precise genomemodification and lower adverse effects or example, the mutation can becorrected by the insertions or deletions that arise due to the NHEJrepair pathway. If the patient's HFE gene has an inserted or deletedbase, a targeted cleavage can result in a NHEJ-mediated insertion ordeletion that restores the frame. Missense mutations can also becorrected through NHEJ-mediated correction using one or more guide RNA.The ability or likelihood of the cut(s) to correct the mutation can bedesigned or evaluated based on the local sequence and micro-homologies.NHEJ can also be used to delete segments of the gene, either directly orby altering splice donor or acceptor sites through cleavage by one gRNAtargeting several locations, or several gRNAs. This may be useful if anamino acid, domain or exon contains the mutations and can be removed orinverted, or if the deletion otherwise restored function to the protein.Pairs of guide strands have been used for deletions and corrections ofinversions.

Alternatively, the donor for correction by HDR contains the correctedsequence with small or large flanking homology arms to allow forannealing. HDR is essentially an error-free mechanism that uses asupplied homologous DNA sequence as a template during DSB repair. Therate of HDR is a function of the distance between the mutation and thecut site so choosing overlapping or nearby target sites is important.Templates can include extra sequences flanked by the homologous regionsor can contain a sequence that differs from the genomic sequence, thusallowing sequence editing.

In addition to correcting mutations by NHEJ or HDR, a range of otheroptions are possible. If there are small or large deletions or multiplemutations, a cDNA can be knocked in that contains the exons affected. Afull length cDNA can be knocked into any “safe harbor”—i.e.,non-deleterious insertion point that is not the HFE gene itself—, withor without suitable regulatory sequences. If this construct isknocked-in near the HFE regulatory elements, it should havephysiological control, similar to the normal gene. Two or more (e.g., apair) nucleases can be used to delete mutated gene regions, though adonor would usually have to be provided to restore function. In thiscase two gRNA and one donor sequence would be supplied.

Provided herein are cellular, ex vivo and in vivo methods for usinggenome engineering tools to create permanent changes to the genomeby: 1) correcting, by insertions or deletions that arise due to the NHEJpathway, one or more mutations within or near the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene, 2)correcting, by HDR, one or more mutations within or near the HFE gene orother DNA sequences that encode regulatory elements of the HFE gene, or3) deletion of the mutant region and/or knocking-in HFE cDNA or minigeneinto the gene locus or a safe harbour locus, such as, e.g., targetingexons 1-2 (exon 1, intron 1, and exon 2) of an AAVS1 (PPP1R12C) gene, anALB gene, an AngptI3 gene, an ApoC3 gene, an ASGR2 gene, a CCR5 gene, aFIX (F9) gene, a G6PC gene, a Gys2 gene, an HGD gene, an Lp(a) gene, aPcsk9 gene, a Serpina1 gene, a TF gene, and/or a TTR gene, or 4)deletion of the mutant region and knocking-in HFE cDNA or a minigene(comprised of one or more exons and introns or natural or syntheticintrons) into the first exon of HGD, leading to disruption of HGDexpression. HGD−/− hepatocytes have proliferation advantage when FAHactivity is absent or reduced. Inhibition of FAH can be achieved bytreatment with shRNA (AAV) targeting FAH, or siRNA (LNP formulated, orconjugate with GaINAc, or with cholesterol) or treatment with CEHPOBA(see e.g., Paulk et al. “In vivo selection of transplanted hepatocytesby pharmacological inhibition of fumarylacetoacetate hydrolase inwild-type mice.” Mol Ther 2012, 20(10):1981-1987). Assessment ofefficiency of HDR mediated knock-in of cDNA into the first exon canutilize cDNA knock-in into “safe harbor” sites such as: single-strandedor double-stranded DNA having homologous arms to one of the followingregions, for example: ApoC3 (chr11:116829908-116833071), AngptI3(chr1:62,597,487-62,606,305), Serpina1 (chr14:94376747-94390692), Lp(a)(chr6:160531483-160664259), Pcsk9 (chr1:55,039,475-55,064,852), FIX(chrX:139,530,736-139,563,458), ALB (chr4:73,404,254-73,421,411), TTR(chr18:31,591,766-31,599,023), TF (chr3:133,661,997-133,779,005), Gys2(chr12:21,536,188-21,604,857), AAVS1(PPP1R12C)(chr19:55,090,912-55,117,599), HGD (chr3:120,628,167-120,682,570), CCR5(chr3:46,370,854-46,376,206), ASGR2 (chr17:7,101,322-7,114,310), G6PC(chr17: 42,900,796-42,914,432), 5′UTR correspondent to ASS1 oralternative 5′ UTR, complete CDS of HFE and 3′ UTR of HFE or modified 3′UTR and at least 80 nt of the first intron, alternatively same DNAtemplate sequence will be delivered by AAV. Both the HDR and knock-instrategies utilize a donor DNA template in Homology-Directed Repair(HDR). HDR in either strategy may be accomplished by making one or moresingle-stranded breaks (SSBs) or one or more double-stranded breaks(DSBs) at specific sites in the genome by using one or moreendonucleases.

Such methods use endonucleases, such as CRISPR-associated (Cas9, Cpf1and the like) nucleases, to permanently delete, insert, edit or correctone or more mutations within or near the genomic locus of the HFE geneor other DNA sequences that encode regulatory elements of the HFE gene.In this way, examples set forth in the present disclosure can help torestore the reading frame or the wild-type sequence of, or otherwisecorrect, the gene with as few as a single treatment (rather than deliverpotential therapies for the lifetime of the patient).

Provided herein are methods for treating a patient with HHC. An aspectof such method is an ex vivo cell-based therapy. For example, a patientspecific induced pluripotent stem cell (iPSC) can be created. Then, thechromosomal DNA of these iPS cells can be edited using the materials andmethods described herein. Next, the genome-edited iPSCs can bedifferentiated into hepatocytes. Finally, the hepatocytes can beimplanted into the patient.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a biopsy of the patient's liver is performed. Then, a liverspecific progenitor cell or primary hepatocyte is isolated from thebiopsied material. Next, the chromosomal DNA of these liver specificprogenitor cells or primary hepatocytes can be edited using thematerials and methods described herein. Finally, the genome-edited liverspecific progenitor cell or primary hepatocyte can be implanted into thepatient.

Yet another aspect of such method is an ex vivo cell-based therapy. Forexample, a mesenchymal stem cell can be isolated from the patient, whichcan be isolated from the patient's bone marrow by performing a biopsy ofthe patient's bone marrow or isolated from peripheral blood. Next, thechromosomal DNA of these mesenchymal stem cells can be edited using thematerials and methods described herein. Next, the genome-editedmesenchymal stem cells can be differentiated into hepatocytes. Finally,these hepatocytes can be implanted into the patient.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. Nuclease-based therapeutics can have some level ofoff-target effects. Performing gene correction ex vivo allows one tocharacterize the corrected cell population prior to implantation. Thepresent disclosure includes sequencing the entire genome of thecorrected cells to ensure that the off-target effects, if any, can be ingenomic locations associated with minimal risk to the patient.Furthermore, populations of specific cells, including clonalpopulations, can be isolated prior to implantation.

Another advantage of ex vivo cell therapy relates to genetic correctionin iPSCs compared to other primary cell sources. iPSCs are prolific,making it easy to obtain the large number of cells that will be requiredfor a cell-based therapy. Furthermore, iPSCs are an ideal cell type forperforming clonal isolations. This allows screening for the correctgenomic correction, without risking a decrease in viability. Incontrast, other potential cell types, such as primary hepatocytes, areviable for only a few passages and difficult to clonally expand. Thus,manipulation of iPSCs for the treatment of HHC can be much easier, andcan shorten the amount of time needed to make the desired geneticcorrection.

Methods can also include an in vivo based therapy. Chromosomal DNA ofthe cells in the patient is edited using the materials and methodsdescribed herein.

An advantage of in vivo gene therapy can be the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient's own cells, which are isolated, manipulated and returned to thesame patient.

Also provided herein is a cellular method for editing the HFE gene in acell by genome editing. For example, a cell can be isolated from apatient or animal. Then, the chromosomal DNA of the cell can be editedusing the materials and methods described herein.

The methods provided herein, regardless of whether a cellular or ex vivoor in vivo method, can involve one or a combination of the following: 1)correcting, by insertions or deletions that arise due to the impreciseNHEJ pathway, one or more mutations within or near the HFE gene or otherDNA sequences that encode regulatory elements of the HFE gene, 2)correcting, by HDR, one or more mutations within or near the HFE gene orother DNA sequences that encode regulatory elements of the HFE gene, or3) deletion of the mutant region and/or knocking-in HFE cDNA into thegene locus or at a heterologous location in the genome (such as a safeharbor locus, such as, e.g., targeting exons 1-2 (exon 1, intron 1, andexon 2) of an AAVS1 (PPP1R12C) gene, an ALB gene, an AngptI3 gene, anApoC3 gene, an ASGR2 gene, a CCR5 gene, a FIX (F9) gene, a G6PC gene, aGys2 gene, an HGD gene, an Lp(a) gene, a Pcsk9 gene, a Serpina1 gene, aTF gene, and/or a TTR gene). Both the HDR and knock-in strategiesutilize a donor DNA template in HDR. HDR in either strategy may beaccomplished by making one or more single-stranded breaks (SSBs) ordouble-stranded breaks (DSBs) at specific sites in the genome by usingone or more endonucleases.

For example, the NHEJ correction strategy can involve correcting aspecific mutation in the HFE gene by inducing one single stranded breakor double stranded break in the gene of interest with one or more CRISPRendonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA), or two ormore single stranded breaks or double stranded breaks in the gene ofinterest with two or more CRISPR endonucleases and two or more sgRNAs.This approach can require development and optimization of sgRNAs anddonor DNA molecules for the major varient of the HFE gene.

For example, the HDR correction strategy can involve correcting aspecific mutation in the HFE gene by inducing one single stranded breakor double stranded break in the gene of interest with one or more CRISPRendonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA), or two ormore single stranded breaks or double stranded breaks in the gene ofinterest with one or more CRISPR endonucleases and two or more gRNAs, inthe presence of a donor DNA template introduced exogenously to directthe cellular DSB response to Homology-Directed Repair (the donor DNAtemplate can be a short single stranded oligonucleotide, a short doublestranded oligonucleotide, a long single or double stranded DNAmolecule). This approach can require development and optimization ofgRNAs and donor DNA molecules for the major varient of the HFE gene.

For example, the knock-in strategy involves knocking-in HFE cDNA intothe locus of the gene using a gRNA (e.g., crRNA+tracrRNA, or sgRNA) or apair of gRNAs targeting upstream of or in the first or other exon and/orintron of the HFE gene, or in a safe harbor site (such as, e.g., exon1-2 of, AAVS1 (PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9),G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and/or TTR). The donor DNAcan be single or double stranded DNA having homologous arms to the human6p21.3 region.

The advantages for the above strategies (correction and knock-in) aresimilar, including in principle both short and long term beneficialclinical and laboratory effects. The knock-in approach does provide oneadvantage over the correction approach—the ability to treat all patientsversus only a subset of patients.

In addition to the above genome editing strategies, another strategyinvolves modulating expression, function, or activity of HFE by editingin the regulatory sequence.

In addition to the editing options listed above, Cas9 or similarproteins can be used to target effector domains to the same target sitesthat can be identified for editing, or additional target sites withinrange of the effector domain. A range of chromatin modifying enzymes,methylases or demethlyases can be used to alter expression of the targetgene. One possibility is increasing the expression of the HFE protein ifthe mutation leads to lower activity. These types of epigeneticregulation have some advantages, particularly as they are limited inpossible off-target effects.

A number of types of genomic target sites can be present in addition tomutations in the coding and splicing sequences.

The regulation of transcription and translation implicates a number ofdifferent classes of sites that interact with cellular proteins ornucleotides. Often the DNA binding sites of transcription factors orother proteins can be targeted for mutation or deletion to study therole of the site, though they can also be targeted to change geneexpression. Sites can be added through non-homologous end joining NHEJor direct genome editing by HDR. Increased use of genome sequencing, RNAexpression and genome-wide studies of transcription factor binding haveincreased our ability to identify how the sites lead to developmental ortemporal gene regulation. These control systems can be direct or caninvolve extensive cooperative regulation that can require theintegration of activities from multiple enhancers. Transcription factorstypically bind 6-12 bp-long degenerate DNA sequences. The low level ofspecificity provided by individual sites suggests that complexinteractions and rules are involved in binding and the functionaloutcome. Binding sites with less degeneracy can provide simpler means ofregulation. Artificial transcription factors can be designed to specifylonger sequences that have less similar sequences in the genome and havelower potential for off-target cleavage. Any of these types of bindingsites can be mutated, deleted or even created to enable changes in generegulation or expression (Canver, M. C. et al., Nature (2015)).

Another class of gene regulatory regions having these features ismicroRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play keyroles in posttranscriptional gene regulation. miRNA can regulate theexpression of 30% of all mammalian protein-encoding genes. Specific andpotent gene silencing by double stranded RNA (RNAi) was discovered, plusadditional small noncoding RNA (Canver, M. C. et al., Nature (2015)).The largest class of noncoding RNAs important for gene silencing aremiRNAs. In mammals, miRNAs are first transcribed as a long RNAtranscripts, which can be separate transcriptional units, part ofprotein introns, or other transcripts. The long transcripts are calledprimary miRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNA can be cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported, into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger guide strand (marked with *),can be functional, but is usually degraded. The mature miRNA tethersRISC to partly complementary sequence motifs in target mRNAspredominantly found within the 3′ untranslated regions (UTRs) andinduces posttranscriptional gene silencing (Bartel, D. P. Cell 136,215-233 (2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510(2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs can be encoded by multiple loci, some ofwhich can be expressed from tandemly co-transcribed clusters. Thefeatures allow for complex regulatory networks with multiple pathwaysand feedback controls. miRNAs can be integral parts of these feedbackand regulatory circuits and can help regulate gene expression by keepingprotein production within limits (Herranz, H. & Cohen, S. M. Genes Dev24, 1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin GenetDev 27, 1-6 (2014)).

miRNA can also be important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also have a strong link to cancer and can play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. MicroRNAs can delicately regulate the balance ofangiogenesis, such that experiments depleting all microRNAs suppressestumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNA can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites may lead to decreased expression ofthe targeted gene, while introducing these sites may increaseexpression.

Individual miRNA can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which can be important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNA could also be inhibited by specific targeting of thespecial loop region adjacent to the palindromic sequence. Catalyticallyinactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. etal., Sci Rep 4, 3943 (2014)). In addition to targeting the miRNA, thebinding sites can also be targeted and mutated to prevent the silencingby miRNA.

Human Cells

For ameliorating HHC, as described and illustrated herein, the principaltargets for gene editing are human cells. For example, in the ex vivomethods, the human cells can be somatic cells, which after beingmodified using the techniques as described, can give rise to hepatocytesor progenitor cells. For example, in the in vivo methods, the humancells can be hepatocytes, or cells from other affected organs.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that can beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Progenitor cells (also referred to as stem cells herein) are capable ofboth proliferation and giving rise to more progenitor cells, these inturn having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one aspect, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell that, itself, is derived from a multipotent cell, andso on. While each of these multipotent cells may be considered stemcells, the range of cell types that each can give rise to may varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity may benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, such as a myocyte, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further.

Induced Pluripotent Stem Cells

The genetically engineered human cells described herein can be inducedpluripotent stem cells (iPSCs). An advantage of using iPSCs is that thecells can be derived from the same subject to which the progenitor cellsare to be administered. That is, a somatic cell can be obtained from asubject, reprogrammed to an induced pluripotent stem cell, and thenre-differentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). Because the progenitors areessentially derived from an autologous source, the risk of engraftmentrejection or allergic response can be reduced compared to the use ofcells from another subject or group of subjects. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one aspect, the stem cells used in the disclosed methods arenot embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. Reprogramming can encompassecomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompasse complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a hematopoietic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)] and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); andreferences cited therein. The production of iPSCs can be achieved by theintroduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it may not be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-3/4 or Pouf51), Soxl, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-3/4, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(I,3-Dioxo-IH,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., Cl-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve not only RT-PCR, but can also include detection ofprotein markers. Intracellular markers may be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Hepatocytes

The genetically engineered human cells described herein can behepatocytes. A hepatocyte is a cell of the main parenchymal tissue ofthe liver. Hepatocytes make up 70-85% of the liver's mass. These cellsare involved in: protein synthesis; protein storage; transformation ofcarbohydrates; synthesis of cholesterol, bile salts and phospholipids;detoxification, modification, and excretion of exogenous and endogenoussubstances; and initiation of formation and secretion of bile.

Although the HFE gene is expressed in various tissues, iron is primarilydeposed in hepatocytes and thus correction of the HFE gene should beprimarily targeted to hepatocytes and the liver.

Creating Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a patient specific iPS cell, patient specific iPS cells, or apatient specific iPS cell line. There are many established methods inthe art for creating patient specific iPS cells, as described inTakahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example,the creating step can comprise: isolating a somatic cell, such as a skincell or fibroblast, from the patient; and introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

Performing a Biopsy or Aspirate of the Patient's Bone Marrow

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicinecan be applied first. A biopsy or aspirate can be performed according toany of the known methods in the art. For example, in a liver biopsy, aneedle is injected into the liver through the skin or the belly,capturing the liver tissue. For example, in a bone marrow aspirate, alarge needle is used to enter the pelvis bone to collect bone marrow.

Isolating a Liver Specific Progenitor Cell or Primary Hepatocyte

Liver specific progenitor cells and primary hepatocytes may be isolatedaccording to any method known in the art. For example, human hepatocytesare isolated from fresh surgical specimens (e.g., an autologous sample).Healthy liver tissue is used to isolate hepatocytes by collagenasedigestion. The obtained cell suspension is filtered through a 100-mmnylon mesh and sedimented by centrifugation at 50 g for 5 minutes,resuspended, and washed two to three times in cold wash medium. Humanliver stem cells are obtained by culturing under stringent conditions ofhepatocytes obtained from fresh liver preparations. Hepatocytes seededon collagen-coated plates are cultured for 2 weeks. After 2 weeks,surviving cells are removed, and characterized for expression of stemcells markers (Herrera et al, STEM CELLS 2006; 24: 2840-2850).

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells can be isolated according to any method known inthe art, such as from a patient's bone marrow or peripheral blood. Forexample, marrow aspirate can be collected into a syringe with heparin.Cells can be washed and centrifuged on a Percoll™ density gradient.Cells, such as blood cells, liver cells, interstitial cells,macrophages, mast cells, and thymocytes, can be separated using densitygradient centrifugation media, Percoll™. The cells can then be culturedin Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C etal., Science 1999; 284:143-147).

Genome Editing

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. Examples of methods of genome editing describedherein include methods of using site-directed nucleases to cutdeoxyribonucleic acid (DNA) at precise target locations in the genome,thereby creating single-strand or double-strand DNA breaks at particularlocations within the genome. Such breaks can be and regularly arerepaired by natural, endogenous cellular processes, such as HDR andNHEJ. These two main DNA repair processes consist of a family ofalternative pathways. NHEJ directly joins the DNA ends resulting from adouble-strand break, sometimes with the loss or addition of nucleotidesequence, which may disrupt or enhance gene expression. HDR utilizes ahomologous sequence, or donor sequence, as a template for inserting adefined DNA sequence at the break point. The homologous sequence can bein the endogenous genome, such as a sister chromatid. Alternatively, thedonor can be an exogenous nucleic acid, such as a plasmid, asingle-strand oligonucleotide, a double-stranded oligonucleotide, aduplex oligonucleotide or a virus, that has regions of high homologywith the nuclease-cleaved locus, but which can also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism canbe microhomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ”, in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few basepairs flanking the DNAbreak site to drive a more favored DNA end joining repair outcome, andrecent reports have further elucidated the molecular mechanism of thisprocess. In some instances it may be possible to predict likely repairoutcomes based on analysis of potential microhomologies at the site ofthe DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of site-directedpolypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introducedouble-strand breaks or single-strand breaks in nucleic acids, e.g.,genomic DNA. The double-strand break can stimulate a cell's endogenousDNA-repair pathways (e.g., homology-dependent repair or non-homologousend joining or alternative non-homologous end joining (A-NHEJ) ormicrohomology-mediated end joining). NHEJ can repair cleaved targetnucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor template cancomprise sequences that can be homologous to sequences flanking thetarget nucleic acid cleavage site. The sister chromatid can be used bythe cell as the repair template. However, for the purposes of genomeediting, the repair template can be supplied as an exogenous nucleicacid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide, double-stranded oligonucleotide, or viral nucleic acid.With exogenous donor templates, an additional nucleic acid sequence(such as a transgene) or modification (such as a single or multiple basechange or a deletion) can be introduced between the flanking regions ofhomology so that the additional or altered nucleic acid sequence alsobecomes incorporated into the target locus. MMEJ can result in a geneticoutcome that is similar to NHEJ in that small deletions and insertionscan occur at the cleavage site. MMEJ can make use of homologoussequences of a few basepairs flanking the cleavage site to drive afavored end-joining DNA repair outcome. In some instances it may bepossible to predict likely repair outcomes based on analysis ofpotential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence or polynucleotide donortemplate) herein. The donor polynucleotide, a portion of the donorpolynucleotide, a copy of the donor polynucleotide, or a portion of acopy of the donor polynucleotide can be inserted into the target nucleicacid cleavage site. The donor polynucleotide can be an exogenouspolynucleotide sequence, i.e., a sequence that does not naturally occurat the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified byendogenous RNaseIII, and then hybridizes to a crRNA repeat in thepre-crRNA array. Endogenous RNaseIII can be recruited to cleave thepre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimmingto produce the mature crRNA form (e.g., 5′ trimming). The tracrRNA canremain hybridized to the crRNA, and the tracrRNA and the crRNA associatewith a site-directed polypeptide (e.g., Cas9). The crRNA of thecrRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleicacid to which the crRNA can hybridize. Hybridization of the crRNA to thetarget nucleic acid can activate Cas9 for targeted nucleic acidcleavage. The target nucleic acid in a Type II CRISPR system is referredto as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacentmotif such that Cpf1-crRNA complexes efficiently cleave target DNApreceeded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpf1 cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpf1 contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG.1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAMsequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed can be administered to a cell or a patientas either: one or more polypeptides, or one or more mRNAs encoding thepolypeptide.

In the context of a CRISPR/Cas or CRISIDR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Casor CRISPR/Cpf1 systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprises a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9”refers to both naturally-occurring and recombinant Cas9s. Cas9 enzymescontemplated herein can comprise a HNH or HNH-like nuclease domain,and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., HDR or NHEJ or alternative non-homologous end joining(A-NHEJ) or microhomology-mediated end joining (MMEJ)). NHEJ can repaircleaved target nucleic acid without the need for a homologous template.This can sometimes result in small deletions or insertions (indels) inthe target nucleic acid at the site of cleavage, and can lead todisruption or alteration of gene expression. HDR can occur when ahomologous repair template, or donor, is available. The homologous donortemplate can comprise sequences that are homologous to sequencesflanking the target nucleic acid cleavage site. The sister chromatid canbe used by the cell as the repair template. However, for the purposes ofgenome editing, the repair template can be supplied as an exogenousnucleic acid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide or viral nucleic acid. With exogenous donor templates,an additional nucleic acid sequence (such as a transgene) ormodification (such as a single or multiple base change or a deletion)can be introduced between the flanking regions of homology so that theadditional or altered nucleic acid sequence also becomes incorporatedinto the target locus. MMEJ can result in a genetic outcome that issimilar to NHEJ in that small deletions and insertions can occur at thecleavage site. MMEJ can make use of homologous sequences of a fewbasepairs flanking the cleavage site to drive a favored end-joining DNArepair outcome. In some instances it may be possible to predict likelyrepair outcomes based on analysis of potential microhomologies in thenuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides. The site-directed polypeptidecan comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids. The site-directed polypeptide cancomprises at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. The site-directed polypeptide cancomprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids in a HNH nuclease domain of thesite-directed polypeptide. The site-directed polypeptide can comprise atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at least: 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at most: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a RuvC nuclease domain of the site-directed polypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild-type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a single-strand break (SSB) on a targetnucleic acid (e.g., by cutting only one of the sugar-phosphate backbonesof a double-strand target nucleic acid). The mutation can result in lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than5%, or less than 1% of the nucleic acid-cleaving activity in one or moreof the plurality of nucleic acid-cleaving domains of the wild-type sitedirected polypeptide (e.g., Cas9 from S. pyogenes, supra). The mutationcan result in one or more of the plurality of nucleic acid-cleavingdomains retaining the ability to cleave the complementary strand of thetarget nucleic acid, but reducing its ability to cleave thenon-complementary strand of the target nucleic acid. The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the non-complementary strand of thetarget nucleic acid, but reducing its ability to cleave thecomplementary strand of the target nucleic acid. For example, residuesin the wild-type exemplary S. pyogenes Cas9 polypeptide, such as Asp10,His840, Asn854 and Asn856, are mutated to inactivate one or more of theplurality of nucleic acid-cleaving domains (e.g., nuclease domains). Theresidues to be mutated can correspond to residues Asp10, His840, Asn854and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide(e.g., as determined by sequence and/or structural alignment).Non-limiting examples of mutations include D10A, H840A, N854A or N856A.One skilled in the art will recognize that mutations other than alaninesubstitutions can be suitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A H840A mutation can be combined with oneor more of D10A, N854A, or N856A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. A N854Amutation can be combined with one or more of H840A, D10A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. A N856A mutation can be combined with one or moreof H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases”.

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagines, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscan comprise a mutation of histidine 840, and wherein the mutationreduces the cleaving activity of the nuclease domain(s) by at least 50%.

The one or more site-directed polypeptides, e.g. DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, caneffect or cause one double-strand break at a specific locus in thegenome.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeatsequence and tracrRNA sequence hybridize to each other to form a duplex.In the Type V guide RNA (gRNA), the crRNA forms a duplex. In bothsystems, the duplex can bind a site-directed polypeptide, such that theguide RNA and site-direct polypeptide form a complex. Thegenome-targeting nucleic acid can provide target specificity to thecomplex by virtue of its association with the site-directed polypeptide.The genome-targeting nucleic acid thus can direct the activity of thesite-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:1-64,980 of the Sequence Listing, shown with the genome location oftheir target sequence and the associated Cas9 cut site, wherein thegenome location is based on the GRCh38/hg38 human genome assembly.

Each guide RNA can be designed to include a spacer sequencecomplementary to its genomic target sequence. For example, each of thespacer sequences in SEQ ID NOs: 1-64,980 of the Sequence Listing can beput into a single RNA chimera or a crRNA (along with a correspondingtracrRNA). See Jinek et al., Science, 337, 816-821 (2012) and Deltchevaet al., Nature, 471, 602-607 (2011).

The genome-targeting nucleic acid can be a double-molecule guide RNA.The genome-targeting nucleic acid can be a single-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. The sgRNA can comprise a less than a 20 nucleotidespacer sequence at the 5′ end of the sgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a variable length spacer sequencewith 17-30 nucleotides at the 5′ end of the sgRNA sequence (see Table1).

The sgRNA can comprise no uracil at the 3′end of the sgRNA sequence,such as in SEQ ID NO. 64,985 of Table 1. The sgRNA can comprise one ormore uracil at the 3′end of the sgRNA sequence, such as in SEQ ID NOs.64,986-64,987 in Table 1. For example, the sgRNA can comprise 1 uracil(U) at the 3′end of the sgRNA sequence. The sgRNA can comprise 2 uracil(UU) at the 3′end of the sgRNA sequence. The sgRNA can comprise 3 uracil(UUU) at the 3′end of the sgRNA sequence. The sgRNA can comprise 4uracil (UUUU) at the 3′end of the sgRNA sequence. The sgRNA can comprise5 uracil (UUUUU) at the 3′end of the sgRNA sequence. The sgRNA cancomprise 6 uracil (UUUUUU) at the 3′end of the sgRNA sequence. The sgRNAcan comprise 7 uracil (UUUUUUU) at the 3′end of the sgRNA sequence. ThesgRNA can comprise 8 uracil (UUUUUUUU) at the 3′end of the sgRNAsequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides. Forexample, modified sgRNAs can comprise 2′-O-methyl phosphorothioatenucleotides as the first 3 nucleotides and 2′-O-methyl phosphorothioatenucleotides as the last 3 nucleotides, such as in SEQ ID NO. 64,984 inTable 1 where “*” indicates 2′-O-methyl phosphorothioate nucleotides.The first 3 nucleotides in the n₍₁₇₋₃₀₎ region of SEQ ID NO. 64,987 canbe 2′-O-methyl phosphorothioate nucleotides (i.e.: n*). The last 3nucleotides within the u₍₁₋₈₎ region of SEQ ID NO. 64,987 can be2′-O-methyl phosphorothioate urasils (i.e.: u*). In certain exampleswhere the u₍₁₋₈₎ region of SEQ ID NO. 64,987 contains less than 3urasil, one or two nucleotides prior to the u₍₁₋₈₎ region can be2′-O-methyl phosphorothioate nucleotides such that the last threenucleotides of the sequence are 2′-O-methyl phosphorothioatenucleotides.

TABLE 1 SEQ ID NO. sgRNA sequence 64,983nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuu gaaaaaguggcaccgagucggugcuuuu 64,984n*n*n*nnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaa cuugaaaaaguggcaccgagucggugcun*n*n*64,985 nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuu gaaaaaguggcaccgagucggugc 64,986n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcu₍₁₋₈₎ 64,987n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcu₍₁₋₈₎

By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system,or other smaller RNAs, can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are morereadily generated enzymatically.

Various types of RNA modifications can be introduced during or afterchemical synthesis and/or enzymatic generation of RNAs, e.g.,modifications that enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on- or off-target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5′ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR/Cas system herein, the spacer sequence can be designed tohybridize to a target nucleic acid that is located 5′ of a PAM of theCas9 enzyme used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target nucleic acid can comprise at most: 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.The target nucleic acid sequence can comprise 20 bases immediately 5′ ofthe first nucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO. 64,981), the target nucleicacid can comprise the sequence that corresponds to the Ns, wherein N isany nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer can comprise 19nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which may bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

A minimum CRISPR repeat sequence can be a sequence with at least about30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or 100% sequence identity toa reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

A minimum CRISPR repeat sequence can comprise nucleotides that canhybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPRrepeat sequence and a minimum tracrRNA sequence can form a duplex, i.e.a base-paired double-stranded structure. Together, the minimum CRISPRrepeat sequence and the minimum tracrRNA sequence can bind to thesite-directed polypeptide. At least a part of the minimum CRISPR repeatsequence can hybridize to the minimum tracrRNA sequence. At least a partof the minimum CRISPR repeat sequence can comprise at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% complementary to theminimum tracrRNA sequence. At least a part of the minimum CRISPR repeatsequence can comprise at most about 30%, about 40%, about 50%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or 100% complementary to the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some examples, the minimum CRISPRrepeat sequence can be approximately 9 nucleotides in length. Theminimum CRISPR repeat sequence can be approximately 12 nucleotides inlength.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence can be atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to a site-directed polypeptide. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5′-XXXY-3′ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5′-AAGY-3′ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge can comprise at most one wobble pairing. A bulge can comprise atleast one purine nucleotide. A bulge can comprise at least 3 purinenucleotides. A bulge sequence can comprises at least 5 purinenucleotides. A bulge sequence can comprise at least one guaninenucleotide. In some examples, a bulge sequence can comprise at least oneadenine nucleotide.

Hairpins

In various examples, one or more hairpins can be located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3′ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3′ tracrRNA Sequence

A 3′ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3′ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3′ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3′ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3′ tracrRNA sequence cancomprise two duplexed regions.

The 3′ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3′ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3′ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3′ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence may be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides. ThetracrRNA extension sequence can have a length of less than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Genome Engineering Strategies to Correct Cells by Deletion, Insertion,or Correction of One or More Mutations within or Near the HFE Gene, orby Knocking-in HFE cDNA into the Locus of the Corresponding HFE Gene orSafe Harbor Site

The methods of the present disclosure can involve correction of one orboth of the mutant alleles. Gene editing to correct the mutation has theadvantage of restoration of correct expression levels and temporalcontrol. Sequencing the patient's HFE alleles allows for design of thegene editing strategy to best correct the identified mutation(s).

A step of the ex vivo methods of the present disclosure can compriseediting/correcting the patient specific iPSC cells using genomeengineering. Alternatively, a step of the ex vivo methods of the presentdisclosure can comprise editing/correcting the liver specific progenitorcell, primary hepatocyte, or mesenchymal stem cell. Likewise, a step ofthe in vivo methods of the present disclosure can compriseediting/correcting the cells in a HHC patient using genome engineering.Similarly, a step in the cellular methods of the present disclosure cancomprise editing/correcting the HFE gene in a human cell by genomeengineering.

HHC patients exhibit one or more mutations in the HFE gene. Therefore,different patients will generally require different correctionstrategies. Any CRISPR endonuclease may be used in the methods of thepresent disclosure, each CRISPR endonuclease having its own associatedPAM, which may or may not be disease specific. For example, gRNA spacersequences for targeting the HFE gene with a CRISPR/Cas9 endonucleasefrom S. pyogenes have been identified in SEQ ID NOs. 1-4,455 of theSequence Listing. gRNA spacer sequences for targeting the HFE gene witha CRISPR/Cas9 endonuclease from S. aureus have been identified in SEQ IDNOs. 4,456-4,979 of the Sequence Listing. gRNA spacer sequences fortargeting the HFE gene with a CRISPR/Cas9 endonuclease from S.thermophilus have been identified in SEQ ID NOs. 4,980-5,139 of theSequence Listing. gRNA spacer sequences for targeting the HFE gene witha CRISPR/Cas9 endonuclease from T. denticola have been identified in SEQID NOs. 5,140-5,192 of the Sequence Listing. gRNA spacer sequences fortargeting the HFE gene with a CRISPR/Cas9 endonuclease from N.meningitides have been identified in SEQ ID NOs. 5,193-5,617 of theSequence Listing. gRNA spacer sequences for targeting the HFE gene witha CRISPR/Cpf1 endonuclease from Acidominococcus, Lachnospiraceae, andFranciscella Novicida have been identified in SEQ ID NOs. 5,618-10,121of the Sequence Listing.

For example, the mutation can be corrected by the insertions ordeletions that arise due to the imprecise NHEJ repair pathway. If thepatient's HFE gene has an inserted or deleted base, a targeted cleavagecan result in a NHEJ-mediated insertion or deletion that restores theframe. Missense mutations can also be corrected through NHEJ-mediatedcorrection using one or more guide RNA. The ability or likelihood of thecut(s) to correct the mutation can be designed or evaluated based on thelocal sequence and micro-homologies. NHEJ can also be used to deletesegments of the gene, either directly or by altering splice donor oracceptor sites through cleavage by one gRNA targeting several locations,or several gRNAs. This may be useful if an amino acid, domain or exoncontains the mutations and can be removed or inverted, or if thedeletion otherwise restored function to the protein. Pairs of guidestrands have been used for deletions and corrections of inversions.

Alternatively, the donor for correction by HDR contains the correctedsequence with small or large flanking homology arms to allow forannealing. HDR is essentially an error-free mechanism that uses asupplied homologous DNA sequence as a template during DSB repair. Therate of HDR is a function of the distance between the mutation and thecut site so choosing overlapping or nearest target sites is important.Templates can include extra sequences flanked by the homologous regionsor can contain a sequence that differs from the genomic sequence, thusallowing sequence editing.

In addition to correcting mutations by NHEJ or HDR, a range of otheroptions are possible. If there are small or large deletions or multiplemutations, a cDNA can be knocked in that contains the exons affected. Afull length cDNA can be knocked into any “safe harbor”, but must use asupplied or other promoter. If this construct is knocked into thecorrect location, it will have physiological control, similar to thenormal gene. Pairs of nucleases can be used to delete mutated generegions, though a donor would usually have to be provided to restorefunction. In this case two gRNA would be supplied and one donorsequence.

Some genome engineering strategies involve correction of one or moremutations in or near the HFE gene, or deleting the mutant HFE DNA and/orknocking-in HFE cDNA into the locus of the corresponding gene or a safeharbor locus by HDR, which is also known as homologous recombination(HR). Homology directed repair can be one strategy for treating patientsthat have one or more mutations in or near the HFE gene. Thesestrategies can restore the HFE gene and reverse, treat, and/or mitigatethe diseased state. These strategies can require a more custom approachbased on the location of the patient's mutation(s). Donor nucleotidesfor correcting mutations often are small (<300 bp). This isadvantageous, as HDR efficiencies may be inversely related to the sizeof the donor molecule. Also, it is expected that the donor templates canfit into size constrained adeno-associated virus (AAV) molecules, whichhave been shown to be an effective means of donor template delivery.

Homology direct repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁶ cells receiving a homologous donor alone. The rate of HDR at aparticular nucleotide is a function of the distance to the cut site, sochoosing overlapping or nearest target sites is important. Gene editingoffers the advantage over gene addition, as correcting in situ leavesthe rest of the genome unperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotermay increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

In addition to wildtype endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection,nano-particle, micro-injection, or viral transduction. A range oftethering options has been proposed to increase the availability of thedonors for HDR. Examples include attaching the donor to the nuclease,attaching to DNA binding proteins that bind nearby, or attaching toproteins that are involved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several nonhomologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as alt-NHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. Maresca,M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23, 539-546 (2013).

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HR. Acombination approach may be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

The HFE gene contains 7 exons. Any one or more of the 7 exons or nearbyintrons can be repaired in order to correct a mutation and restore HFEprotein activity. Alternatively, there are various mutations associatedwith HHC, which are a combination of insertions, deletions, missense,nonsense, frameshift and other mutations, with the common effect ofinactivating the HFE gene. Any one or more of the mutations can berepaired in order to restore the inactive HFE gene expression. Forexample, one or more of the following pathological variants may becorrected: C282Y, H63D, S65C, or combinations thereof (See Table 2). Asa further alternative, HFE cDNA can be knocked-in to the locus of thecorresponding gene or knocked-in to a safe harbor site, such as AAVS1.In some examples, the methods can provide one gRNA or a pair of gRNAsthat can be used to facilitate incorporation of a new sequence from apolynucleotide donor template to correct one or more mutations or toknock-in a part of or the entire HFE gene or cDNA.

TABLE 2 Variant Location Variant type C282Y 6:26092913, rs1800562missense H63D 6:26090951, rs1799945 missense S65C 6:26090957, rs1800730missense

The methods can provide gRNA pairs that make a deletion by cutting thegene twice, one gRNA cutting at the 5′ end of one or more mutations andthe other gRNA cutting at the 3′ end of one or more mutations thatfacilitates insertion of a new sequence from a polynucleotide donortemplate to replace the one or more mutations. The cutting can beaccomplished by a pair of DNA endonucleases that each makes a DSB in thegenome, or by multiple nickases that together make a DSB in the genome.

Alternatively, the methods can provide one gRNA to make onedouble-strand cut around one or more mutations that facilitatesinsertion of a new sequence from a polynucleotide donor template toreplace the one or more mutations. The double-strand cut can be made bya single DNA endonuclease or multiple nickases that together make a DSBin the genome.

Illustrative modifications within the HFE gene include replacementswithin or near (proximal) to the mutations referred to above, such aswithin the region of less than 3 kb, less than 2 kb, less than 1 kb,less than 0.5 kb upstream or downstream of the specific mutation. Giventhe relatively wide variations of mutations in the HFE gene, it will beappreciated that numerous variations of the replacements referencedabove (including without limitation larger as well as smallerdeletions), would be expected to result in restoration of the HFEprotein activity.

Such variants can include replacements that are larger in the 5′ and/or3′ direction than the specific mutation in question, or smaller ineither direction. Accordingly, by “near” or “proximal” with respect tospecific replacements, it is intended that the SSB or DSB locusassociated with a desired replacement boundary (also referred to hereinas an endpoint) can be within a region that is less than about 3 kb fromthe reference locus noted. The SSB or DSB locus can be more proximal andwithin 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb. In the caseof small replacement, the desired endpoint can be at or “adjacent to”the reference locus, by which it is intended that the endpoint can bewithin 100 bp, within 50 bp, within 25 bp, or less than about 10 bp to 5bp from the reference locus.

Examples comprising larger or smaller replacements can be expected toprovide the same benefit, as long as the HFE protein activity isrestored. It is thus expected that many variations of the replacementsdescribed and illustrated herein can be effective for ameliorating HHC.

Another genome engineering strategy involves exon deletion. Targeteddeletion of specific exons can be an attractive strategy for treating alarge subset of patients with a single therapeutic cocktail. Deletionscan either be single exon deletions or multi-exon deletions. Whilemulti-exon deletions can reach a larger number of patients, for largerdeletions the efficiency of deletion greatly decreases with increasedsize. Therefore, deletions range can be from 40 to 10,000 base pairs(bp) in size. For example, deletions can range from 40-100; 100-300;300-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; or5,000-10,000 base pairs in size.

As stated previously, the HFE gene contains 7 exons. Any one or more ofthe 7 exons, or aberrant intronic splice acceptor or donor sites, may bedeleted in order to restore the HFE reading frame. In some embodiments,the methods provide gRNA pairs that can be used to delete exons 1, 2, 3,4, 5, 6, 7 or any combinations thereof.

In order to ensure that the pre-mRNA is properly processed followingdeletion, the surrounding splicing signals can be deleted. Splicingdonor and acceptors are generally within 100 base pairs of theneighboring intron. Therefore, in some examples, methods can provide allgRNAs that cut approximately +/−100-3100 bp with respect to eachexon/intron junction of interest.

For any of the genome editing strategies, gene editing can be confirmedby sequencing or PCR analysis.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first nonlimiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another nonlimiting example of target sequence selection oroptimization, the frequency of off-target activity for a particularcombination of target sequence and gene editing endonuclease (i.e. thefrequency of DSBs occurring at sites other than the selected targetsequence) can be assessed relative to the frequency of on-targetactivity. In some cases, cells that have been correctly edited at thedesired locus can have a selective advantage relative to other cells.Illustrative, but nonlimiting, examples of a selective advantage includethe acquisition of attributes such as enhanced rates of replication,persistence, resistance to certain conditions, enhanced rates ofsuccessful engraftment or persistence in vivo following introductioninto a patient, and other attributes associated with the maintenance orincreased numbers or viability of such cells. In other cases, cells thathave been correctly edited at the desired locus can be positivelyselected for by one or more screening methods used to identify, sort orotherwise select for cells that have been correctly edited. Bothselective advantage and directed selection methods can take advantage ofthe phenotype associated with the correction. In some cases, cells canbe edited two or more times in order to create a second modificationthat creates a new phenotype that is used to select or purify theintended population of cells. Such a second modification could becreated by adding a second gRNA for a selectable or screenable marker.In some cases, cells can be correctly edited at the desired locus usinga DNA fragment that contains the cDNA and also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off-target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but can also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs can be regularly induced andrepaired in normal cells. During repair, the original sequence can bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten basepairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce replacementsthat result in restoration of HFE protein activity, as well as theselection of specific target sequences within such regions that aredesigned to minimize off-target events relative to on-target events.

Nucleic Acid Modifications

In some cases, polynucleotides introduced into cells can comprise one ormore modifications that can be used individually or in combination, forexample, to enhance activity, stability or specificity, alter delivery,reduce innate immune responses in host cells, or for other enhancements,as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9/Cpf1 system to edit anyone or more genomic loci.

Using the CRISPR/Cas9/Cpf1 system for purposes of nonlimitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9/Cpf1 genomeediting complex comprising guide RNAs, which can be single-moleculeguides or double-molecule, and a Cas or Cpf1 endonuclease. Modificationsof guide RNAs can also or alternatively be used to enhance theinitiation, stability or kinetics of interactions between the genomeediting complex with the target sequence in the genome, which can beused, for example, to enhance on-target activity. Modifications of guideRNAs can also or alternatively be used to enhance specificity, e.g., therelative rates of genome editing at the on-target site as compared toeffects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas orCpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNAses present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9/Cpf1, for example, one ormore types of modifications can be made to guide RNAs (including thoseexemplified above), and/or one or more types of modifications can bemade to RNAs encoding Cas endonuclease (including those exemplifiedabove).

By way of illustration, guide RNAs used in the CRISPR/Cas9/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means,enabling a number of modifications to be readily incorporated, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach that can be used for generatingchemically-modified RNAs of greater length is to produce two or moremolecules that are ligated together. Much longer RNAs, such as thoseencoding a Cas9 endonuclease, are more readily generated enzymatically.While fewer types of modifications are available for use inenzymatically produced RNAs, there are still modifications that can beused to, e.g., enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as describedfurther below and in the art; and new types of modifications areregularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl,or 2′-fluoro-modified nucleotide. In some examples, RNA modificationscan comprise 2′-fluoro, 2′-amino or 2′ O-methyl modifications on theribose of pyrimidines, abasic residues, or an inverted base at the 3′end of the RNA. Such modifications can be routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH,˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone), CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones[see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂, or O(CH₂)n CH₃, where n is from 1 to about10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; ON; CF₃, OCF₃; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications can also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides can also have sugarmimetics, such as cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-am inohexyl)adenine, and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, pp 75-77 (1980); Gebeyehu et al., Nucl. Acids Res.15:4513 (1997). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke,S. T. and Lebleu, B., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are aspects of basesubstitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspectsof base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and US Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but arenot limited to, lipid moieties such as a cholesterol moiety, cholicacid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, analiphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See,e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs (such asm7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013), and references cited therein.

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

RNPs

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thepre-complexed material can then be administered to a cell or a patient.Such pre-complexed material is known as a ribonucleoprotein particle(RNP).

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Additional vectors contemplated for eukaryotic target cells include, butare not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3. Othervectors can be used so long as they are compatible with the host cell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy-Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. As described in the art, a variety of targetingmoieties can be used to enhance the preferential interaction of suchvehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, can be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, can be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be generally combined in a 1:1 molarratio. Alternatively, the endonuclease, crRNA and tracrRNA can begenerally combined in a 1:1:1 molar ratio. However, a wide range ofmolar ratios can be used to produce a RNP.

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12,AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in,for example, international patent application publication number WO01/83692. See Table 3.

TABLE 3 AAV Serotype Genbank Accession No. AAV-1 NC_002077.1 AAV-2NC_001401.2 AAV-3 NC_001729.1 AAV-3B AF028705.1 AAV-4 NC_001829.1 AAV-5NC_006152.1 AAV-6 AF028704.1 AAV-7 NC_006260.1 AAV-8 NC_006261.1 AAV-9AX753250.1 AAV-10 AY631965.1 AAV-11 AY631966.1 AAV-12 DQ813647.1 AAV-13EU285562.1

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line can then be infected with ahelper virus, such as adenovirus. The advantages of this method are thatthe cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus, rather than plasmids, to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mo1. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others. See Table 4.

TABLE 4 Tissue/Cell Type Serotype Liver AAV8, AA3, AA5, AAV9 Skeletalmuscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV5, AAV1,AAV4 RPE AAV5, AAV4 Photoreceptor cells AAV5 Lung AAV9 Heart AAV8Pancreas AAV8 Kidney AAV2, AAV8

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplexvirus.

In some cases, Cas9 mRNA, sgRNA targeting one or two loci in the HFEgene, and donor DNA can each be separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle.

In some cases, Cas9 mRNA can be formulated in a lipid nanoparticle,while sgRNA and donor DNA can be delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nano-particles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas9/Cpf1 system). In some ex vivo examples herein, thegenetically modified cell can be genetically modified progenitor cell.In some in vivo examples herein, the genetically modified cell can be agenetically modified liver cell. A genetically modified cell comprisingan exogenous genome-targeting nucleic acid and/or an exogenous nucleicacid encoding a genome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of the HFE gene orprotein expression or activity, for example Western Blot analysis of theHFE protein or quantifying HFE mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratinghereditary haemochromatosis.

The term “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Differentiation of Genome-Edited iPSCs into Hepatocytes

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSCs into hepatocytes. Thedifferentiating step can be performed according to any method known inthe art. For example, hiPSC are differentiated into definitive endodermusing various treatments, including activin and B27 supplement (LifeTechnology). The definitive endoderm is further differentiated intohepatocyte, the treatment includes: FGF4, HGF, BMP2, BMP4, Oncostatin M,Dexametason, etc (Duan et al, STEM CELLS; 2010; 28:674-686, Ma et al,STEM CELLS TRANSLATIONAL MEDICINE 2013; 2:409-419).

Differentiation of Genome-Edited Mesenchymal Stem Cells into Hepatocytes

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem cells intohepatocytes. The differentiating step can be performed according to anymethod known in the art. For example, hMSC are treated with variousfactors and hormones, including insulin, transferrin, FGF4, HGF, bileacids (Sawitza I et al, Sci Rep. 2015; 5: 13320).

Implanting Cells into Patients

Another step of the ex vivo methods of the present disclosure cancomprise implanting liver specific progenitor cells or primaryhepatocytes into patients. This implanting step can be accomplishedusing any method of implantation known in the art. For example, thegenetically modified cells can be injected directly in the patient'sliver or otherwise administered to the patient. The genetically modifiedcells may be purified ex vivo using a selected marker.

Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein can involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Administration & Efficacy

The terms “administering,” “introducing” and “transplanting” are usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny, can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of myogenic progenitor cells is administered via asystemic route of administration, such as an intraperitoneal orintravenous route.

The terms “individual”, “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of HHC, e.g., priorto the development of abdominal pain, weakness, lethargy, and weightloss, cirrhosis, progressive increase in skin pigmentation, diabetesmellitus, congestive heart failure, and/or arrhythmias, arthritis, andhypogonadism. Accordingly, the prophylactic administration of a liverprogenitor cell population serves to prevent hereditaryhaemochromatosis.

When provided therapeutically, liver progenitor cells are provided at(or after) the onset of a symptom or indication of hereditaryhaemochromatosis, e.g., upon the onset of disease.

The liver progenitor cell population being administered according to themethods described herein can comprise allogeneic liver progenitor cellsobtained from one or more donors. “Allogeneic” refers to a liverprogenitor cell or biological samples comprising liver progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, a liverprogenitor cell population being administered to a subject can bederived from one more unrelated donor subjects, or from one or morenon-identical siblings. In some cases, syngeneic liver progenitor cellpopulations can be used, such as those obtained from geneticallyidentical animals, or from identical twins. The liver progenitor cellscan be autologous cells; that is, the liver progenitor cells areobtained or isolated from a subject and administered to the samesubject, i.e., the donor and recipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of HHC, and relates to a sufficientamount of a composition to provide the desired effect, e.g., to treat asubject having HHC. The term “therapeutically effective amount”therefore refers to an amount of progenitor cells or a compositioncomprising progenitor cells that is sufficient to promote a particulareffect when administered to a typical subject, such as one who has or isat risk for HHC. An effective amount would also include an amountsufficient to prevent or delay the development of a symptom of thedisease, alter the course of a symptom of the disease (for example butnot limited to, slow the progression of a symptom of the disease), orreverse a symptom of the disease. It is understood that for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5×10²progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

Modest and incremental increases in the levels of functional HFEexpressed in cells of patients having HHC can be beneficial forameliorating one or more symptoms of the disease, for increasinglong-term survival, and/or for reducing side effects associated withother treatments. Upon administration of such cells to human patients,the presence of liver progenitors that are producing increased levels offunctional HFE is beneficial. In some cases, effective treatment of asubject gives rise to at least about 3%, 5% or 7% functional HFErelative to total HFE in the treated subject. In some examples,functional HFE will be at least about 10% of total HFE. In someexamples, functional HFE will be at least about 20% to 30% of total HFE.Similarly, the introduction of even relatively limited subpopulations ofcells having significantly elevated levels of functional HFE can bebeneficial in various patients because in some situations normalizedcells will have a selective advantage relative to diseased cells.However, even modest levels of liver progenitors with elevated levels offunctional HFE can be beneficial for ameliorating one or more aspects ofHHC in patients. In some examples, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or moreof the liver progenitors in patients to whom such cells are administeredare producing increased levels of functional HFE.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time. Modes of administration includeinjection, infusion, instillation, or ingestion. “Injection” includes,without limitation, intravenous, intramuscular, intra-arterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. In someexamples, the route is intravenous. For the delivery of cells,administration by injection or infusion can be made.

The cells can be administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof HHC can be determined by the skilled clinician. However, a treatmentis considered “effective treatment,” if any one or all of the signs orsymptoms of, as but one example, levels of functional HFE are altered ina beneficial manner (e.g., increased by at least 10%), or otherclinically accepted symptoms or markers of disease are improved orameliorated. Efficacy can also be measured by failure of an individualto worsen as assessed by hospitalization or need for medicalinterventions (e.g., progression of the disease is halted or at leastslowed). Methods of measuring these indicators are known to those ofskill in the art and/or described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibiting thedisease, e.g., arresting, or slowing the progression of symptoms; or (2)relieving the disease, e.g., causing regression of symptoms; and (3)preventing or reducing the likelihood of the development of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with HHC by increasing the amount of functionalHFE in the individual. Early signs typically associated with HHC includefor example, abdominal pain, weakness, lethargy, and weight loss,cirrhosis, progressive increase in skin pigmentation, diabetes mellitus,congestive heart failure, and/or arrhythmias, arthritis, andhypogonadism.

Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, (2) the site-directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide; and (2) a reagent forreconstitution and/or dilution of the vector.

In any of the above kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of theabove kits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the above kits, the kit can further comprise a polynucleotideto be inserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit described above can further comprise one or more additionalreagents, where such additional reagents are selected from a buffer, abuffer for introducing a polypeptide or polynucleotide into a cell, awash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. A kit can also comprise one or more components that can be used tofacilitate or enhance the on-target binding or the cleavage of DNA bythe endonuclease, or improve the specificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or subpackaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g. via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Other Possible Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage alsorequires an adjacent motif, the PAM, which differs between differentCRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NGGPAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMsincluding NNNNGATT, NNNNNGTTT and NNNNGCTT. A number of other Cas9orthologs target protospacer adjacent to alternative PAMs.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nulceases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the FokI nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single basepair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the FokI domain to reduceoff-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9/Cpf1 “nickase”mutants in which one of the Cas9 cleavage domains has been deactivated.DNA nicks can be used to drive genome editing by HDR, but at lowerefficiency than with a DSB. The main benefit is that off-target nicksare quickly and accurately repaired, unlike the DSB, which is prone toNHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res.39(14):6315-25(2011); Weber et al., PLoS One. 6(2):e16765 (2011); Wanget al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); andCermak T et al., Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO. 64,982), GIY-YIG, His-Cis box, H—N—H, PD-(D/E)xK,and Vsr-like that are derived from a broad range of hosts, includingeukarya, protists, bacteria, archaea, cyanobacteria and phage.

As with ZFNs and TALENs, HEs can be used to create a DSB at a targetlocus as the initial step in genome editing. In addition, some naturaland engineered HEs cut only a single strand of DNA, thereby functioningas site-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012); and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-Tevl (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-FokI or dCpf1-Fok1 and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a FokI domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). BecauseFokI must dimerize to become catalytically active, two guide RNAs arerequired to tether two FokI fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-Tevl, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-Tevl, with the expectation that off-targetcleavage can be further reduced.

Methods and Compositions of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting inventions: In a first method, Method 1, thepresent disclosure provides a method for editing an HFE gene in a humancell by genome editing, the method comprising: introducing into thehuman cell one or more deoxyribonucleic acid (DNA) endonucleases toeffect one or more single-strand breaks (SSBs) or double-strand breaks(DSBs) within or near the HFE gene or other DNA sequences that encoderegulatory elements of the HFE gene that results in a permanentdeletion, insertion, correction, or modulation of expression or functionof one or more mutations within or near or affecting the expression orfunction of the HFE gene and results in restoration of HFE proteinactivity.

In another method, Method 2, the present disclosure provides an ex vivomethod for inserting a haemochromatosis (HFE) gene in a human cell bygenome editing, the method comprising: introducing into the human cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near a safe harbor locus that results in a permanent insertion of theHFE gene, and results in restoration of HFE protein activity.

In another method, Method 3, the present disclosure provides an ex vivomethod for treating a patient with HHC, the method comprising: creatinga patient specific induced pluripotent stem cell (iPSC); editing withinor near an HFE gene or other DNA sequences that encode regulatoryelements of the HFE gene of the iPSC, or editing within or near a safeharbor locus of the iPSC; differentiating the genome-edited iPSC into ahepatocyte; and implanting the hepatocyte into the patient.

In another method, Method 4, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 3 whereinthe creating step comprises: isolating a somatic cell from the patient;and introducing a set of pluripotency-associated genes into the somaticcell to induce the somatic cell to become a pluripotent stem cell.

In another method, Method 5, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 4, whereinthe somatic cell is a fibroblast.

In another method, Method 6, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Methods 4 or 5,wherein the set of pluripotency-associated genes is one or more of thegenes selected from the group consisting of OCT4, SOX2, KLF4, Lin28,NANOG and cMYC.

In another method, Method 7, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods3-6, wherein the editing step comprises introducing into the iPSC one ormore deoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene that results in a permanent deletion, insertion,correction, or modulation of expression or function of one or moremutations within or near or affecting the expression or function of theHFE gene, or within or near a safe harbor locus, that results inrestoration of HFE protein activity.

In another method, Method 8, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 7, whereinthe safe harbor locus is selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 9, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods3-8, wherein the differentiating step comprises contacting thegenome-edited iPSC with one or more of activin, B27 supplement, FGF4,HGF, BMP2, BMP4, Oncostatin M, or Dexametason.

In another method, Method 10, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods3-9, wherein the implanting step comprises implanting the hepatocyteinto the patient by transplantation, local injection, systemic infusion,or combinations thereof.

In another method, Method 11, the present disclosure provides an ex vivomethod for treating a patient with HHC, the method comprising:performing a biopsy of the patient's liver; isolating a liver specificprogenitor cell or primary hepatocyte from the patient's liver; editingwithin or near an HFE gene or other DNA sequences that encode regulatoryelements of the HFE gene of the liver specific progenitor cell orprimary hepatocyte or editing within or near a safe harbor locus of theliver specific progenitor cell or primary hepatocyte; and implanting thegenome-edited liver specific progenitor cell or primary hepatocyte intothe patient.

In another method, Method 12, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 11, whereinthe isolating step comprises: perfusion of fresh liver tissues withdigestion enzymes, cell differencial centrifugation, cell culturing, orcombinations thereof.

In another method, Method 13, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Methods 11 or 12,wherein the editing step comprises introducing into the liver specificprogenitor cell or primary hepatocyte one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene or within ornear a safe harbor locus that results in a permanent deletion,insertion, correction, or modulation of expression or function of one ormore mutations within or near or affecting the expression or function ofthe HFE gene, or within or near a safe harbor locus, and restoration ofHFE protein activity.

In another method, Method 14, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 13, whereinthe safe harbor locus is selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 15, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods11-14, wherein the implanting step comprises implanting thegenome-edited liver specific progenitor cell or primary hepatocyte intothe patient by transplantation, local injection, systemic infusion, orcombinations thereof.

In another method, Method 16, the present disclosure provides an ex vivomethod for treating a patient with HHC, the method comprising: isolatinga mesenchymal stem cell from the patient; editing within or near an HFEgene or other DNA sequences that encode regulatory elements of the HFEgene of the mesenchymal stem cell, or editing within or near a safeharbor locus of the mesenchymal stem cell; differentiating thegenome-edited mesenchymal stem cell into a hepatocyte; and implantingthe hepatocyte into the patient.

In another method, Method 17, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 16, whereinthe mesenchymal stem cell is isolated from the patient's bone marrow byperforming a biopsy of the patient's bone marrow or the mesenchymal stemcell is isolated from peripheral blood.

In another method, Method 18, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Methods 16 or 17,wherein the isolating step comprises: aspiration of bone marrow andisolation of mesenchymal cells using density gradient centrifugationmedia.

In another method, Method 19, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods16-18, wherein the editing step comprises introducing into themesenchymal stem cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene that resultsin a permanent deletion, insertion, correction, or modulation ofexpression or function of one or more mutations within or near oraffecting the expression or function of the HFE gene or within or near asafe harbor locus, that results in restoration of HFE protein activity.

In another method, Method 20, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in Method 19, whereinthe safe harbor locus is selected from the group consisting of AAVS1(PPP1R12C), ALB, AngptI3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 21, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods16-20, wherein the differentiating step comprises contacting thegenome-edited mesenchymal stem cell with one or more of insulin,transferrin, FGF4, HGF, or bile acids.

In another method, Method 22, the present disclosure provides an ex vivomethod for treating a patient with HHC as provided in any one of Methods16-21, wherein the implanting step comprises implanting the hepatocyteinto the patient by transplantation, local injection, systemic infusion,or combinations thereof.

In another method, Method 23, the present disclosure provides an in vivomethod for treating a patient with HHC, the method comprising the stepof editing an HFE gene in a cell of the patient or other DNA sequencesthat encode regulatory elements of the HFE gene, or editing within ornear a safe harbor locus in a cell of the patient.

In another method, Method 24, the present disclosure provides an in vivomethod for treating a patient with HHC as provided in Method 23, whereinthe editing step comprises introducing into the cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene that results in a permanent deletion, insertion,correction, or modulation of expression or function of one or moremutations within or near or affecting the expression or function of theHFE gene or within or near a safe harbor locus that results inrestoration of HFE protein activity.

In another method, Method 25, the present disclosure provides a methodaccording to any one of Methods 1, 2, 7, 13, 19, and 24, wherein the oneor more DNA endonucleases is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1endonuclease; a homolog thereof, a recombination of the naturallyoccurring molecule thereof, codon-optimized thereof, or modifiedversions thereof, and combinations thereof.

In another method, Method 26, the present disclosure provides a methodas provided in Method 25, wherein the method comprises introducing intothe cell one or more polynucleotides encoding the one or more DNAendonucleases.

In another method, Method 27, the present disclosure provides a methodas provided in Method 25, wherein the method comprises introducing intothe cell one or more ribonucleic acids (RNAs) encoding the one or moreDNA endonucleases.

In another method, Method 28, the present disclosure provides a methodas provided in Methods 26 or 27, wherein the one or more polynucleotidesor one or more RNAs is one or more modified polynucleotides or one ormore modified RNAs.

In another method, Method 29, the present disclosure provides a methodas provided in Method 25, wherein the DNA endonuclease is one or moreproteins or polypeptides.

In another method, Method 30, the present disclosure provides a methodas provided in any one of Methods 1-29, wherein the method furthercomprises introducing into the cell one or more guide ribonucleic acids(gRNAs).

In another method, Method 31, the present disclosure provides a methodas provided in Method 30, wherein the one or more gRNAs aresingle-molecule guide RNA (sgRNAs).

In another method, Method 32, the present disclosure provides a methodas provided in Methods 30 or 31, wherein the one or more gRNAs or one ormore sgRNAs is one or more modified gRNAs or one or more modifiedsgRNAs.

In another method, Method 33, the present disclosure provides a methodas provided in any one of Methods 29-31, wherein the one or more DNAendonucleases is pre-complexed with one or more gRNAs or one or moresgRNAs.

In another method, Method 34, the present disclosure provides a methodas provided in any one of Methods 1-33, wherein the method furthercomprises introducing into the cell a polynucleotide donor templatecomprising at least a portion of the wild-type HFE gene, DNA sequencesthat encode wild-type regulatory elements of the HFE gene, or cDNA.

In another method, Method 35, the present disclosure provides a methodas provided in Method 34, wherein the at least a portion of thewild-type HFE gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5,exon 6, exon 7, intronic regions, fragments or combinations thereof, orthe entire HFE gene or cDNA.

In another method, Method 36, the present disclosure provides a methodas provided in any one of Methods 34 or 35, wherein the donor templateis either single or double stranded.

In another method, Method 37, the present disclosure provides a methodas provided in any one of Methods 34-36, wherein the donor template hashomologous arms to the 6p21.3 region.

In another method, Method 38, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24, wherein themethod further comprises introducing into the cell one guide ribonucleicacid (gRNA) and a polynucleotide donor template comprising at least aportion of the wild-type HFE gene, and wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect onesingle-strand break (SSB) or double-strand break (DSB) at a locus withinor near the HFE gene or other DNA sequences that encode regulatoryelements of the HFE gene, or within or near a safe harbor locus, thatfacilitates insertion of a new sequence from the polynucleotide donortemplate into the chromosomal DNA at the locus or safe harbor locus thatresults in a permanent insertion or correction of a part of thechromosomal DNA of the HFE gene or other DNA sequences that encoderegulatory elements of the HFE gene proximal to the locus, or safeharbor locus, and wherein the gRNA comprises a spacer sequence that iscomplementary to a segment of the locus, or safe harbor locus.

In another method, Method 39, the present disclosure provides a methodas provided in Method 38, wherein proximal means nucleotides bothupstream and downstream of the locus or safe harbor locus.

In another method, Method 40, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24, wherein themethod further comprises introducing into the cell one or more guideribonucleic acid (gRNAs) and a polynucleotide donor template comprisingat least a portion of the wild-type HFE gene, and wherein the one ormore DNA endonucleases is one or more Cas9 or Cpf1 endonucleases thateffect a pair of single-strand breaks (SSBs) or double-strand breaks(DSBs), the first at a 5′ locus and the second at a 3′ locus, within ornear the HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene or within or near a safe harbor locus that facilitatesinsertion of a new sequence from the polynucleotide donor template intothe chromosomal DNA between the 5′ locus and the 3′ locus that resultsin a permanent insertion or correction of the chromosomal DNA betweenthe 5′ locus and the 3′ locus within or near the HFE gene or other DNAsequences that encode regulatory elements of the HFE gene or within ornear a safe harbor locus, and wherein the first guide RNA comprises aspacer sequence that is complementary to a segment of the 5′ locus andthe second guide RNA comprises a spacer sequence that is complementaryto a segment of the 3′ locus.

In another method, Method 41, the present disclosure provides a methodas provided in any one of Methods 38-40, wherein the one or more gRNAsare one or more single-molecule guide RNA (sgRNAs).

In another method, Method 42, the present disclosure provides a methodas provided in any one of Methods 38-41, wherein the one or more gRNAsor one or more sgRNAs is one or more modified gRNAs or one or moremodified sgRNAs.

In another method, Method 43, the present disclosure provides a methodas provided in any one of Methods 38-42, wherein the one or more DNAendonucleases is pre-complexed with one or more gRNAs or one or moresgRNAs.

In another method, Method 44, the present disclosure provides a methodas provided in any one of Methods 38-43, wherein the at least a portionof the wild-type HFE gene or cDNA is exon 1, exon 2, exon 3, exon 4,exon 5, exon 6, exon 7, intronic regions, fragments or combinationsthereof, or the entire HFE gene or cDNA.

In another method, Method 45, the present disclosure provides a methodas provided in any one of Methods 38-44, wherein the donor template iseither a single or double stranded polynucleotide.

In another method, Method 46, the present disclosure provides a methodas provided in any one of Methods 38-45, wherein the donor template hashomologous arms to the 6p21.3 region.

In another method, Method 47, the present disclosure provides a methodas provided in any one of Method 44, wherein the SSB or DSB are in thefirst, second, third, fourth, fifth, sixth, seventh exon, orcombinations thereof, of the HFE gene.

In another method, Method 48, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, 24, 30-33, or 41-43,wherein the gRNA or sgRNA is directed to one or more of the followingpathological variants: C282Y, H63D, S65C, or combinations thereof.

In another method, Method 49, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24-48, whereinthe insertion or correction is by HDR.

In another method, Method 50, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24, wherein themethod further comprises introducing into the cell two guide ribonucleicacid (gRNAs), and wherein the one or more DNA endonucleases is one ormore Cas9 or Cpf1 endonucleases that effect a pair of double-strandbreaks (DSBs), the first at a 5′ DSB locus and the second at a 3′ DSBlocus, within or near the HFE gene or other DNA sequences that encoderegulatory elements of the HFE gene, or within or near a safe harborlocus that causes a deletion of the chromosomal DNA between the 5′ DSBlocus and the 3′ DSB locus that results in a permanent deletion of thechromosomal DNA between the 5′ DSB locus and the 3′ DSB locus within ornear the HFE gene or other DNA sequences that encode regulatory elementsof the HFE gene or safe harbor locus, and wherein the first guide RNAcomprises a spacer sequence that is complementary to a segment of the 5′DSB locus and the second guide RNA comprises a spacer sequence that iscomplementary to a segment of the 3′ DSB locus.

In another method, Method 51, the present disclosure provides a methodas provided in Method 50, wherein the two gRNAs are two single-moleculeguide RNA (sgRNAs).

In another method, Method 52, the present disclosure provides a methodas provided in Methods 50 or 51 wherein the two gRNAs or two sgRNAs aretwo modified gRNAs or two modified sgRNAs.

In another method, Method 53, the present disclosure provides a methodas provided in any one of Methods 50-52, wherein the one or more DNAendonucleases is pre-complexed with one or two gRNAs or one or twosgRNAs.

In another method, Method 54, the present disclosure provides a methodas provided in any one of Methods 50-53, wherein both the 5′ DSB and 3′DSB are in or near either the first exon, first intron, second exon,second intron, third exon, third intron, fourth exon, fourth intron,fifth exon, fifth intron, sixth exon, sixth intron, seventh exon,seventh intron, or combinations thereof, of the HFE gene.

In another method, Method 55, the present disclosure provides a methodas provided in any one of Methods 50-54, wherein the deletion is adeletion of 1 kb or less.

In another method, Method 56, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24-55 wherein theCas9 or Cpf1 mRNA, gRNA, and donor template are either each formulatedinto separate lipid nanoparticles or all co-formulated into a lipidnanoparticle.

In another method, Method 57, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24-55, whereinthe Cas9 or Cpf1 mRNA is formulated into a lipid nanoparticle, and boththe gRNA and donor template are delivered to the cell by anadeno-associated virus (AAV) vector.

In another method, Method 58, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24-55, whereinthe Cas9 or Cpf1 mRNA is formulated into a lipid nanoparticle, and thegRNA is delivered to the cell by electroporation and donor template isdelivered to the cell by an adeno-associated virus (AAV) vector.

In another method, Method 59, the present disclosure provides a methodas provided in any one of Methods 1-58, wherein the HFE gene is locatedon Chromosome 6: 26087458-26095569 (Genome ReferenceConsortium—GRCh38/hg38).

In another method, Method 60, the present disclosure provides a methodas provided in any one of Methods 1, 2, 7, 13, 19, and 24, wherein therestoration of HFE protein activity is compared to wild-type or normalHFE protein activity.

In a first composition, Composition 1, the present disclosure providesone or more guide ribonucleic acids (gRNAs) for editing an HFE gene in acell from a patient with HHC, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 1-64,980 of the Sequence Listing.

In another composition, Composition 2, the present disclosure providesthe one or more gRNAs of Composition 1, wherein the one or more gRNAsare one or more single-molecule guide RNAs (sgRNAs).

In another composition, Composition 3, the present disclosure providesthe one or more gRNAs or sgRNAs of Compositions 1 or 2, wherein the oneor more gRNAs or one or more sgRNAs is one or more modified gRNAs or oneor more modified sgRNAs.

In another composition, Composition 4, the present disclosure providesthe one or more gRNAs or sgRNAs of Compositions 1-3, wherein the cell isselected from a group consisting of a liver cell, skin cell, pancreaticcell, heart cell, joint cell, or cell from the testes.

In another method, Method 65, the present disclosure provides a methodas provided in any one of Method 1, wherein the human cell is selectedfrom a group consisting of a liver cell, skin cell, pancreatic cell,heart cell, joint cell, or cell from the testes.

In another method, Method 66, the present disclosure provides a methodas provided in any one of Method 23, wherein the human cell is selectedfrom a group consisting of a liver cell, skin cell, pancreatic cell,heart cell, joint cell, or cell from the testes.

Definitions

The term “comprising” or “comprises” is used in reference tocompositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the aspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements, termed “genomic modifications”herein, in the HFE gene that lead to permanent correction of mutationsin the genomic locus, or expression at a heterologous locus, thatrestore HFE protein activity.

Single gRNAs spanning different regions of the HFE gene were selectedand tested for cutting efficiencies (Table 5).

TABLE 5 SEQ gRNA gRNA Sequence PAM ID NO. Name without PAM Sequence 409Hfe_T5 CGCTTGCTGCGTGAGTCCGA GGG 4117 Hfe_T338 AGATGCCCAGTAAAACTTCC TGG377 Hfe_T252 TGAGCCTAGGCAATAGCTGT AGG 378 Hfe_T101 GAGCCTAGGCAATAGCTGTAGGG 4114 Hfe_T394 TACAGCTATTGCCTAGGCTC AGG 4112 Hfe_T240TCACCCTACAGCTATTGCCT AGG 379 Hfe_T158 AATAGCTGTAGGGTGACTTC TGG 4106Hfe_T377 TTTTGGGGGGCGGGGAAACG GGG 4102 Hfe_T278 CTCCGCTTCTTTTGGGGGGC GGG4100 Hfe_T402 AAATCTCCGCTTCTTTTGGG GGG 4099 Hfe_T285TAAATCTCCGCTTCTTTTGG GGG 4098 Hfe_T398 TTAAATCTCCGCTTCTTTTG GGG 386Hfe_T238 AAAAGAAGCGGAGATTTAAC GGG 4097 Hfe_T304 GTTAAATCTCCGCTTCTTTT GGG387 Hfe_T88 AAAGAAGCGGAGATTTAACG GGG 4096 Hfe_T108 CGTTAAATCTCCGCTTCTTTTGG 388 Hfe_T18 GGAGATTTAACGGGGACGTG CGG 391 Hfe_T229CGGGGACGTGCGGCCAGAGC TGG 398 Hfe_T60 GGGAAATGGGCCCGCGAGCC AGG 399Hfe_T117 AATGGGCCCGCGAGCCAGGC CGG 4093 Hfe_T99 GAAGCGCCGGCCTGGCTCGC GGG4092 Hfe_T100 AGAAGCGCCGGCCTGGCTCG CGG 4091 Hfe_T239CAGGAGGAGAAGCGCCGGCC TGG 401 Hfe_T118 CCTGATGCTTTTGCAGACCG CGG 4087Hfe_T246 CGGTCTGCAAAAGCATCAGG AGG 4085 Hfe_T24 CCGCGGTCTGCAAAAGCATC AGG403 Hfe_T27 TTTGCAGACCGCGGTCCTGC AGG 404 Hfe_T34 TTGCAGACCGCGGTCCTGCAGGG 405 Hfe_T146 TGCAGACCGCGGTCCTGCAG GGG 4082 Hfe_T152CAAGCGCCCCTGCAGGACCG CGG 4081 Hfe_T78 CACGCAGCAAGCGCCCCTGC AGG 408Hfe_T11 GCGCTTGCTGCGTGAGTCCG AGG 410 Hfe_T269 CTGCGTGAGTCCGAGGGCTG CGG411 Hfe_T157 TGCGTGAGTCCGAGGGCTGC GGG 413 Hfe_T19 CCGAGGGCTGCGGGCGAACTAGG 414 Hfe_T1 CGAGGGCTGCGGGCGAACTA GGG 415 Hfe_T3 GAGGGCTGCGGGCGAACTAGGGG 4077 Hfe_T61 CCTAGTTCGCCCGCAGCCCT CGG 416 Hfe_T28CTGCGGGCGAACTAGGGGCG CGG 417 Hfe_T68 CGGGCGAACTAGGGGCGCGG CGG 418Hfe_T122 GGGCGAACTAGGGGCGCGGC GGG 419 Hfe_T113 GGCGAACTAGGGGCGCGGCG GGG420 Hfe_T138 GCGAACTAGGGGCGCGGCGG GGG 423 Hfe_T274 ACTAGCTTTTTCTTTGCGCTTGG 426 Hfe_T208 GCTTGGGAGTTTGCTAACTT TGG 428 Hfe_T264TGGGAGTTTGCTAACTTTGG AGG 3678 Hfe_T131 CTCATACCATCAGCTGTGTC TGG 964Hfe_T132 CTGATGGTATGAGTTGATGC AGG 969 Hfe_T207 CCTCCTACTACACATGGTTA AGG3671 Hfe_T352 TTAACCATGTGTAGTAGGAG GGG 3670 Hfe_T233CTTAACCATGTGTAGTAGGA GGG 3669 Hfe_T171 CCTTAACCATGTGTAGTAGG AGG 3667Hfe_T59 AGGCCTTAACCATGTGTAGT AGG 3659 Hfe_T385 TAGTGCAGAGAGTGTGAACC TGG972 Hfe_T547 CTCTCTGCACTACCTCTTCA TGG 973 Hfe_T303 TCTCTGCACTACCTCTTCATGGG 3654 Hfe_T448 GCTCTGAGGCACCCATGAAG AGG 977 Hfe_T458CTTCATGGGTGCCTCAGAGC AGG 978 Hfe_T622 GGTGCCTCAGAGCAGGACCT TGG 3651Hfe_T667 AAGACCAAGGTCCTGCTCTG AGG 980 Hfe_T732 TCTTTCCTTGTTTGAAGCTT TGG981 Hfe_T740 CTTTCCTTGTTTGAAGCTTT GGG 3646 Hfe_T281 GTAGCCCAAAGCTTCAAACAAGG 982 Hfe_T44 GTTTGAAGCTTTGGGCTACG TGG 3642 Hfe_T102GATCATAGAACACGAACAGC TGG 986 Hfe_T4 TGATCATGAGAGTCGCCGTG TGG 988Hfe_T106 GTGTGGAGCCCCGAACTCCA TGG 989 Hfe_T33 TGTGGAGCCCCGAACTCCAT GGG3639 Hfe_T151 ATGGAGTTCGGGGCTCCACA CGG 3638 Hfe_T91 CTGGAAACCCATGGAGTTCGGGG 3637 Hfe_T388 ACTGGAAACCCATGGAGTTC GGG 3636 Hfe_T234TACTGGAAACCCATGGAGTT CGG 3634 Hfe_T552 GAAATTCTACTGGAAACCCA TGG 3633Hfe_T372 CATCTGGCTTGAAATTCTAC TGG 3632 Hfe_T494 GACTCAGCTGCAGCCACATC TGG1000 Hfe_T694 CAGCTGAGTCAGAGTCTGAA AGG 1002 Hfe_T762TGAGTCAGAGTCTGAAAGGG TGG 1003 Hfe_T579 GAGTCAGAGTCTGAAAGGGT GGG 1004Hfe_T558 ACATGTTCACTGTTGACTTC TGG 1005 Hfe_T397 TGTTGACTTCTGGACTATTA TGG1010 Hfe_T608 ACAACCACAGCAAGGGTATG TGG 1013 Hfe_T566CACAGCAAGGGTATGTGGAG AGG 1014 Hfe_T721 ACAGCAAGGGTATGTGGAGA GGG 3624Hfe_T374 CTCTCCACATACCCTTGCTG TGG 1015 Hfe_T687 CAGCAAGGGTATGTGGAGAG GGG3621 Hfe_T411 AAGCTCTGACAACCTCAGGA AGG 3619 Hfe_T530TGAAAAGCTCTGACAACCTC AGG 1028 Hfe_T429 GCTGGAAGTCTGAGGTCTTG TGG 1029Hfe_T386 CTGGAAGTCTGAGGTCTTGT GGG 1032 Hfe_T441 GTCTGAGGTCTTGTGGGAGC AGG1033 Hfe_T636 TCTGAGGTCTTGTGGGAGCA GGG 1041 Hfe_T658ATTTGCTTCCTGAGATCATT TGG 1042 Hfe_T359 TCCTGAGATCATTTGGTCCT TGG 1043Hfe_T290 CCTGAGATCATTTGGTCCTT GGG 1044 Hfe_T346 CTGAGATCATTTGGTCCTTG GGG3610 Hfe_T168 CCCAAGGACCAAATGATCTC AGG 1045 Hfe_T446GATCATTTGGTCCTTGGGGA TGG 1049 Hfe_T553 CCTTGGGGATGGTGGAAATA GGG 1050Hfe_T216 GAAATAGGGACCTATTCCTT TGG 1053 Hfe_T376 TCCTTTGGTTGCAGTTAACA AGG1054 Hfe_T188 TTGGTTGCAGTTAACAAGGC TGG 3604 Hfe_T232GCCTTGTTAACTGCAACCAA AGG 1055 Hfe_T230 TGGTTGCAGTTAACAAGGCT GGG 1056Hfe_T329 GGTTGCAGTTAACAAGGCTG GGG 3601 Hfe_T537 ACCTGCAGGGTGTGGGACTC TGG1062 Hfe_T396 CACACCCTGCAGGTCATCCT GGG 3598 Hfe_T450ACAGCCCAGGATGACCTGCA GGG 1067 Hfe_T48 GCAAGAAGACAACAGTACCG AGG 1068Hfe_T175 CAAGAAGACAACAGTACCGA GGG 1069 Hfe_T84 ACAACAGTACCGAGGGCTAC TGG1071 Hfe_T89 ACCGAGGGCTACTGGAAGTA CGG 1072 Hfe_T50 CCGAGGGCTACTGGAAGTACGGG 3592 Hfe_T144 CCCGTACTTCCAGTAGCCCT CGG 1073 Hfe_T57TACTGGAAGTACGGGTATGA TGG 1074 Hfe_T23 ACTGGAAGTACGGGTATGAT GGG 1076Hfe_T66 GAAGTACGGGTATGATGGGC AGG 3588 Hfe_T504 GTGTGTCAGGGCAGAATTCA AGG1077 Hfe_T521 TGAATTCTGCCCTGACACAC TGG 1078 Hfe_T206TCTGCCCTGACACACTGGAT TGG 3585 Hfe_T404 CTCTCCAATCCAGTGTGTCA GGG 3584Hfe_T211 GCTCTCCAATCCAGTGTGTC AGG 1084 Hfe_T531 ATTGGAGAGCAGCAGAACCC AGG1088 Hfe_T542 CAGGGCCTGGCCCACCAAGC TGG 1090 Hfe_T472CCTGGCCCACCAAGCTGGAG TGG 1091 Hfe_T375 CTGGCCCACCAAGCTGGAGT GGG 3577Hfe_T508 CTTTCCCACTCCAGCTTGGT GGG 1096 Hfe_T528 GTGGGAAAGGCACAAGATTC GGG1098 Hfe_T179 AAAGGCACAAGATTCGGGCC AGG 1101 Hfe_T277AGATTCGGGCCAGGCAGAAC AGG 1102 Hfe_T318 GATTCGGGCCAGGCAGAACA GGG 1103Hfe_T313 CAGGCAGAACAGGGCCTACC TGG 3573 Hfe_T405 CAGGTAGGCCCTGTTCTGCC TGG3572 Hfe_T493 AGGGCAGTCCCTCTCCAGGT AGG 1121 Hfe_T342AGAGGTGTTTTGGACCAACA AGG 1122 Hfe_T104 TGTTTTGGACCAACAAGGTA TGG 1123Hfe_T299 TTTGGACCAACAAGGTATGG TGG 3561 Hfe_T82 GTGTTTCCACCATACCTTGT TGG1125 Hfe_T98 CTTCTGCCCCTATACTCTAG TGG 1128 Hfe_T355 CCTATACTCTAGTGGCAGAGTGG 3558 Hfe_T120 ACTCTGCCACTAGAGTATAG GGG 3557 Hfe_T111CACTCTGCCACTAGAGTATA GGG 3556 Hfe_T205 CCACTCTGCCACTAGAGTAT AGG 1137Hfe_T210 GTTGCAGGGCACGGAATCCC TGG 1138 Hfe_T112 CAGGGCACGGAATCCCTGGT TGG1142 Hfe_T418 ATCCCTGGTTGGAGTTTCAG AGG 3552 Hfe_T327CACCTCTGAAACTCCAACCA GGG 1158 Hfe_T294 ATGAGACAGCCACAAGTCAT GGG 1161Hfe_T204 TCTCCATGCATATGGCTCAA AGG 1162 Hfe_T231 CTCCATGCATATGGCTCAAA GGG3531 Hfe_T322 TTCCCTTTGAGCCATATGCA TGG 1164 Hfe_T361GGCTCAAAGGGAAGTGTCTA TGG 1223 Hfe_T129 TCAGCTATCATATGAATACC AGG 3479Hfe_T250 CCTCACTTGATATTTTGTCC TGG 3478 Hfe_T369 GATTCTTCTACTCTGATAAG TGG1232 Hfe_T8 TCAGAGTAGAAGAATCCTTT AGG 3471 Hfe_T217 AAGAAGCGGACTTGTAAGATAGG 1241 Hfe_T62 AATGCCTCCTAGGTTGACCC AGG 3465 Hfe_T147TTCACCTGGGTCAACCTAGG AGG 3463 Hfe_T289 AGTTTCACCTGGGTCAACCT AGG 3461Hfe_T421 ACAGATGGTCAGTTTCACCT GGG 3460 Hfe_T209 TACAGATGGTCAGTTTCACC TGG3450 Hfe_T311 GACTCTAACACAGTGTCACT TGG 1258 Hfe_T92 CTGTGTTAGAGTCCAATCTTAGG 3448 Hfe_T224 ACCATTTTGTGTCCTAAGAT TGG 1266 Hfe_T193TCCTTCCTCCAACCTATAGA AGG 3438 Hfe_T235 CACTTCCTTCTATAGGTTGG AGG 3433Hfe_T243 TTTACCCTTGCCAGGAAGAC TGG 3431 Hfe_T83 GGGATCTGTTTACCCTTGCC AGG3417 Hfe_T93 CACCAAAGGAGGCACTTGAC AGG 1278 Hfe_T371 TCAAGTGCCTCCTTTGGTGAAGG 3395 Hfe_T198 TCGAACTCCTTGGCATCCAT TGG 3394 Hfe_T186GTCTTTAGGTTCGAACTCCT TGG 1295 Hfe_T41 CCTAAAGACGTATTGCCCAA TGG 1296Hfe_T36 CTAAAGACGTATTGCCCAAT GGG 1297 Hfe_T133 TAAAGACGTATTGCCCAATG GGG3393 Hfe_T39 CCATTGGGCAATACGTCTTT AGG 1298 Hfe_T87 GACGTATTGCCCAATGGGGATGG 1299 Hfe_T45 ACGTATTGCCCAATGGGGAT GGG 1301 Hfe_T801CAATGGGGATGGGACCTACC AGG 1302 Hfe_T125 AATGGGGATGGGACCTACCA GGG 3391Hfe_T366 TGGTAGGTCCCATCCCCATT GGG 3390 Hfe_T167 CTGGTAGGTCCCATCCCCAT TGG1303 Hfe_T324 GGGATGGGACCTACCAGGGC TGG 1304 Hfe_T134CTACCAGGGCTGGATAACCT TGG 3389 Hfe_T174 CAAGGTTATCCAGCCCTGGT AGG 3387Hfe_T140 CAGCCAAGGTTATCCAGCCC TGG 1305 Hfe_T85 ATAACCTTGGCTGTACCCCC TGG1306 Hfe_T155 TAACCTTGGCTGTACCCCCT GGG 1307 Hfe_T242AACCTTGGCTGTACCCCCTG GGG 3385 Hfe_T331 TTCCCCAGGGGGTACAGCCA AGG 3382Hfe_T829 TATCTCTGCTCTTCCCCAGG GGG 3381 Hfe_T731 ATATCTCTGCTCTTCCCCAG GGG

All tested gRNAs can be used for an HDR/correction based editingapproach. Single gRNAs targeting the splice acceptors can be used toinduce exon skipping to restore the reading frame of the HFE gene.Selected pairs of gRNAs can be used to make deletions in the HFE genethat restore the reading frame. Selected pairs of gRNAs can be used tomake deletions that simulate patient mutations and can be used togenerate model HFE mutant lines.

Various Cas orthologs were evaluated for cutting. SP, NM, ST, SA, and TDgRNAs were delivered as RNA, expressed from the U6 promoter in plasmids,or expressed from the U6 promoter in lentivirus. The corresponding Casprotein was either knocked into the cell line of interest andconstitutively expressed, delivered as mRNA, or delivered as protein.The activity of the gRNAs in all the above mentioned formats wereevaluated using TIDE analysis or next generation sequencing in HEK293Tcells, K562 cells, or induced pluripotent stem cells (iPSCs).

Overall, it was determined that most gRNAs tested induced cutting.However, the amount of cutting was highly dependent on the Cas proteintested. It was found that, generally, SP Cas9 gRNAs induce the highestlevels of cutting. Generally, it is beneficial to select gRNAs fortherapeutic application that have the highest cutting efficiencypossible. However, for an iPSC based therapy, the cutting efficiency isnot as important. iPSCs are highly proliferative and make it simple toisolate a clonal population of cells with the desired edit, even whenthe editing efficiency is less than 10%.

Introduction of the defined therapeutic modifications described aboverepresents a novel therapeutic strategy for the potential ameliorationof HHC, as described and illustrated herein.

Example 1—CRISPR/SpCas9 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 1-4,455 of the Sequence Listing.

Example 2—CRISPR/SaCas9 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 4,456-4,979 of the Sequence Listing.

Example 3—CRISPR/StCas9 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 4,980-5,139 of the Sequence Listing.

Example 4—CRISPR/TdCas9 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 24 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 5,140-5,192 of the Sequence Listing.

Example 5—CRISPR/NmCas9 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasgRNA 24 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 5,193-5,617 of the Sequence Listing.

Example 6—CRISPR/Cpf1 Target Sites for the HFE Gene

Regions of the HFE gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 20-24 bp spacer sequences corresponding to the PAM were identified,as shown in SEQ ID NOs: 5,618-10,121 of the Sequence Listing.

Example 7—CRISPR/SpCas9 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence NRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 10,122-12,153 of the SequenceListing.

Example 8—CRISPR/SaCas9 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence NNGRRT. gRNA 20 bp spacer sequences corresponding to the PAMwere identified, as shown in SEQ ID NOs: 12,154-12,324 of the SequenceListing.

Example 9—CRISPR/StCas9 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence NNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAMwere identified, as shown in SEQ ID NOs: 12,325-12,342 of the SequenceListing.

Example 10—CRISPR/TdCas9 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence NAAAAC. gRNA 20 bp spacer sequences corresponding to the PAMwere identified, as shown in SEQ ID NOs: 12,343-12,351 of the SequenceListing.

Example 11—CRISPR/NmCas9 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence NNNNGHTT. gRNA 20 bp spacer sequences corresponding to the PAMwere identified, as shown in SEQ ID NOs: 12,352-12,426 of the SequenceListing.

Example 12—CRISPR/Cpf1 Target Sites for the AAVS1 (PPP1R12C) Gene

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.Each area was scanned for a protospacer adjacent motif (PAM) having thesequence YTN. gRNA 22 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 12,427-13,602 of the SequenceListing.

Example 13—CRISPR/SpCas9 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 13,603-13,770 of the Sequence Listing.

Example 14—CRISPR/SaCas9 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 13,771-13,798 of the SequenceListing.

Example 15—CRISPR/StCas9 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW.

gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 13,799-13,816 of the Sequence Listing.

Example 16—CRISPR/TdCas9 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 13,817-13,821 of the SequenceListing.

Example 17—CRISPR/NmCas9 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasSEQ ID NOs: 13,822-13,845 of the Sequence Listing.

Example 18—CRISPR/Cpf1 Target Sites for the ALB Gene

Exons 1-2 of the ALB gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 13,846-14,224 of the Sequence Listing.

Example 19—CRISPR/SpCas9 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 14,225-14,569 of the SequenceListing.

Example 20—CRISPR/SaCas9 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 14,570-14,605 of the SequenceListing.

Example 21—CRISPR/StCas9 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 14,606-14,628 of the SequenceListing.

Example 22—CRISPR/TdCas9 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 14,629-14,641 of the SequenceListing.

Example 23—CRISPR/NmCas9 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas SEQ ID NOs: 14,642-14,704 of the Sequence Listing.

Example 24—CRISPR/Cpf1 Target Sites for the AngptI3 Gene

Exons 1-2 of the AngptI3 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceYTN. gRNA 22 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 14,705-15,552 of the SequenceListing.

Example 25—CRISPR/SpCas9 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 15,553-15,955 of the Sequence Listing.

Example 26—CRISPR/SaCas9 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 15,956-15,980 of the SequenceListing.

Example 27—CRISPR/StCas9 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 15,981-15,983 of the SequenceListing.

Example 28—CRISPR/TdCas9 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 15,984-15,985 of the SequenceListing.

Example 29—CRISPR/NmCas9 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasSEQ ID NOs: 15,986-15,997 of the Sequence Listing.

Example 30—CRISPR/Cpf1 Target Sites for the ApoC3 Gene

Exons 1-2 of the ApoC3 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 15,998-16,229 of the Sequence Listing.

Example 31—CRISPR/SpCas9 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 16,230-17,997 of the Sequence Listing.

Example 32—CRISPR/SaCas9 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 17,998-18,203 of the SequenceListing.

Example 33—CRISPR/StCas9 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 18,204-18,227 of the SequenceListing.

Example 34—CRISPR/TdCas9 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 18,228-18,239 of the SequenceListing.

Example 35—CRISPR/NmCas9 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasSEQ ID NOs: 18,240-18,322 of the Sequence Listing.

Example 36—CRISPR/Cpf1 Target Sites for the ASGR2 Gene

Exons 1-2 of the ASGR2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 18,323-19,762 of the Sequence Listing.

Example 37—CRISPR/SpCas9 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 19,763-19,965 of the Sequence Listing.

Example 38—CRISPR/SaCas9 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 19,966-19,997 of the SequenceListing.

Example 39—CRISPR/StCas9 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 19,998-20,011 of the SequenceListing.

Example 40—CRISPR/TdCas9 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 20,012-20,013 of the SequenceListing.

Example 41—CRISPR/NmCas9 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasSEQ ID NOs: 20,014-20,041 of the Sequence Listing.

Example 42—CRISPR/Cpf1 Target Sites for the CCR5 Gene

Exons 1-2 of the CCR5 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 20,042-20,341 of the Sequence Listing.

Example 43—CRISPR/SpCas9 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 20,342-21,807 of the SequenceListing.

Example 44—CRISPR/SaCas9 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 21,808-21,970 of the SequenceListing.

Example 45—CRISPR/StCas9 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 21,971-22,031 of the SequenceListing.

Example 46—CRISPR/TdCas9 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 22,032-22,056 of the SequenceListing.

Example 47—CRISPR/NmCas9 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGHTT. SEQ ID NOs: 22,057-22,209 of the Sequence Listing.

Example 48—CRISPR/Cpf1 Target Sites for the FIX (F9) Gene

Exons 1-2 of the FIX (F9) gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceYTN. gRNA 22 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 22,210-24,350 of the SequenceListing.

Example 49—CRISPR/SpCas9 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 24,351-25,366 of the Sequence Listing.

Example 50—CRISPR/SaCas9 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 25,367-25,483 of the SequenceListing.

Example 51—CRISPR/StCas9 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 25,484-25,507 of the SequenceListing.

Example 52—CRISPR/TdCas9 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 25,508-25,516 of the SequenceListing.

Example 53—CRISPR/NmCas9 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasSEQ ID NOs: 25,517-25,606 of the Sequence Listing.

Example 54—CRISPR/Cpf1 Target Sites for the G6PC Gene

Regions of the G6PC gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 25,607-26,701 of the Sequence Listing.

Example 55—CRISPR/SpCas9 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 26,702-32,194 of the Sequence Listing.

Example 56—CRISPR/SaCas9 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 32,195-32,870 of the SequenceListing.

Example 57—CRISPR/StCas9 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 32,871-33,148 of the SequenceListing.

Example 58—CRISPR/TdCas9 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 33,149-33,262 of the SequenceListing.

Example 59—CRISPR/NmCas9 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasSEQ ID NOs: 33,263-33,942 of the Sequence Listing.

Example 60—CRISPR/Cpf1 Target Sites for the Gys2 Gene

Exons 1-2 of the Gys2 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 33,943-42,374 of the Sequence Listing.

Example 61—CRISPR/SpCas9 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 42,375-44,067 of the Sequence Listing.

Example 62—CRISPR/SaCas9 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 44,068-44,281 of the SequenceListing.

Example 63—CRISPR/StCas9 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 44,282-44,364 of the SequenceListing.

Example 64—CRISPR/TdCas9 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 44,365-44,383 of the SequenceListing.

Example 65—CRISPR/NmCas9 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasSEQ ID NOs: 44,384-44,584 of the Sequence Listing.

Example 66—CRISPR/Cpf1 Target Sites for the HGD Gene

Exons 1-2 of the HGD gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 44,585-46,909 of the Sequence Listing.

Example 67—CRISPR/SpCas9 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 46,910-50,704 of the Sequence Listing.

Example 68—CRISPR/SaCas9 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 50,705-51,114 of the SequenceListing.

Example 69—CRISPR/StCas9 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 51,115-51,250 of the SequenceListing.

Example 70—CRISPR/TdCas9 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 51,251-51,285 of the SequenceListing.

Example 71—CRISPR/NmCas9 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasSEQ ID NOs: 51,286-51,653 of the Sequence Listing.

Example 72—CRISPR/Cpf1 Target Sites for the Lp(a) Gene

Exons 1-2 of the Lp(a) gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 51,654-56,274 of the Sequence Listing.

Example 73—CRISPR/SpCas9 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 56,275-58,294 of the Sequence Listing.

Example 74—CRISPR/SaCas9 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 58,295-58,481 of the SequenceListing.

Example 75—CRISPR/StCas9 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 58,482-58,517 of the SequenceListing.

Example 76—CRISPR/TdCas9 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 58,518-58,531 of the SequenceListing.

Example 77—CRISPR/NmCas9 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasSEQ ID NOs: 58,532-58,671 of the Sequence Listing.

Example 78—CRISPR/Cpf1 Target Sites for the PCSK9 Gene

Exons 1-2 of the PCSK9 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 58,672-60,465 of the Sequence Listing.

Example 79—CRISPR/SpCas9 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 60,466-61,603 of the SequenceListing.

Example 80—CRISPR/SaCas9 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,604-61,696 of the SequenceListing.

Example 81—CRISPR/StCas9 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,697-61,708 of the SequenceListing.

Example 82—CRISPR/TdCas9 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,709-61,711 of the SequenceListing.

Example 83—CRISPR/NmCas9 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGHTT. SEQ ID NOs: 61,712-61,762 of the Sequence Listing.

Example 84—CRISPR/Cpf1 Target Sites for the Serpina1 Gene

Exons 1-2 of the Serpina1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceYTN. gRNA 22 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,763-62,566 of the SequenceListing.

Example 85—CRISPR/SpCas9 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 62,567-63,398 of the Sequence Listing.

Example 86—CRISPR/SaCas9 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 63,399-63,484 of the SequenceListing.

Example 87—CRISPR/StCas9 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 63,485-63,496 of the SequenceListing.

Example 88—CRISPR/TdCas9 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 63,497-63,503 of the SequenceListing.

Example 89—CRISPR/NmCas9 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasSEQ ID NOs: 63,504-63,547 of the Sequence Listing.

Example 90—CRISPR/Cpf1 Target Sites for the TF Gene

Exons 1-2 of the TF gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 63,548-64,183 of the Sequence Listing.

Example 91—CRISPR/SpCas9 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 64,184-64,483 of the Sequence Listing.

Example 92—CRISPR/SaCas9 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 64,484-64,524 of the SequenceListing.

Example 93—CRISPR/StCas9 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 64,525-64,541 of the SequenceListing.

Example 94—CRISPR/TdCas9 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 64,542-64,543 of the SequenceListing.

Example 95—CRISPR/NmCas9 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasSEQ ID NOs: 64,544-64,578 of the Sequence Listing.

Example 96—CRISPR/Cpf1 Target Sites for the TTR Gene

Exons 1-2 of the TTR gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 22 bp spacer sequences corresponding to the PAM were identified, asshown in SEQ ID NOs: 64,579-64,980 of the Sequence Listing.

Example 97—Bioinformatics Analysis of the Guide Strands

Candidate guides will be screened and selected in a multi-step processthat involves both theoretical binding and experimentally assessedactivity. By way of illustration, candidate guides having sequences thatmatch a particular on-target site, such as a site within the HFE gene,with adjacent PAM can be assessed for their potential to cleave atoff-target sites having similar sequences, using one or more of avariety of bioinformatics tools available for assessing off-targetbinding, as described and illustrated in more detail below, in order toassess the likelihood of effects at chromosomal positions other thanthose intended. Candidates predicted to have relatively lower potentialfor off-target activity can then be assessed experimentally to measuretheir on-target activity, and then off-target activities at varioussites. Preferred guides have sufficiently high on-target activity toachieve desired levels of gene editing at the selected locus, andrelatively lower off-target activity to reduce the likelihood ofalterations at other chromosomal loci. The ratio of on-target tooff-target activity is often referred to as the “specificity” of aguide.

For initial screening of predicted off-target activities, there are anumber of bioinformatics tools known and publicly available that can beused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9/Cpf1 nuclease system is driven byWatson-Crick base pairing between complementary sequences, the degree ofdissimilarity (and therefore reduced potential for off-target binding)is essentially related to primary sequence differences: mismatches andbulges, i.e. bases that are changed to a non-complementary base, andinsertions or deletions of bases in the potential off-target siterelative to the target site. An exemplary bioinformatics tool calledCOSMID (CRISPR Off-target Sites with Mismatches, Insertions andDeletions) (available on the web at crispr.bme.gatech.edu) compiles suchsimilarities. Other bioinformatics tools include, but are not limitedto, GUIDO, autoCOSMID, and CCtop.

Bioinformatics were used to minimize off-target cleavage in order toreduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof high off-target activity due to nonspecific hybridization of theguide strand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it isimportant to have a bioinformatics tool that can identify potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.The bioinformatics-based tool, COSMID (CRISPR Off-target Sites withMismatches, Insertions and Deletions) was therefore used to searchgenomes for potential CRISPR off-target sites (available on the web atcrispr.bme.gatech.edu). COSMID output ranked lists of the potentialoff-target sites based on the number and location of mismatches,allowing more informed choice of target sites, and avoiding the use ofsites with more likely off-target cleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that may be used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors areweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

Example 98—Testing of Preferred Guides in Cells for On-Target Activity

The gRNAs predicted to have the lowest off-target activity will then betested for on-target activity in a model cell line, such as Huh-7 cells,and evaluated for indel frequency using TIDE or next generationsequencing. TIDE is a web tool to rapidly assess genome editing byCRISPR-Cas9 of a target locus determined by a guide RNA (gRNA or sgRNA).Based on quantitative sequence trace data from two standard capillarysequencing reactions, the TIDE software quantifies the editing efficacyand identifies the predominant types of insertions and deletions(indels) in the DNA of a targeted cell pool. See Brinkman et al, Nucl.Acids Res. (2014) for a detailed explanation and examples.Next-generation sequencing (NGS), also known as high-throughputsequencing, is the catch-all term used to describe a number of differentmodern sequencing technologies including: Illumina (Solexa) sequencing,Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiDsequencing. These recent technologies allow one to sequence DNA and RNAmuch more quickly and cheaply than the previously used Sangersequencing, and as such have revolutionised the study of genomics andmolecular biology.

Transfection of tissue culture cells, allows screening of differentconstructs and a robust means of testing activity and specificity.Tissue culture cell lines, such as Huh-7 cells, are easily transfectedand result in high activity. These or other cell lines will be evaluatedto determine the cell lines that match with hepatocytes and provide thebest surrogate. These cells will then be used for many early stagetests. For example, individual gRNAs for S. pyogenes Cas9 will betransfected into the cells using plasmids, such as, for example, CTx-1,CTx-2, or CTx-3, which are suitable for expression in human cells.Several days later, the genomic DNA is harvested and the target siteamplified by PCR. The cutting activity can be measured by the rate ofinsertions, deletions and mutations introduced by NHEJ repair of thefree DNA ends. Although this method cannot differentiate correctlyrepaired sequences from uncleaved DNA, the level of cutting can begauged by the amount of mis-repair. Off-target activity can be observedby amplifying identified putative off-target sites and using similarmethods to detect cleavage. Translocation can also be assayed usingprimers flanking cut sites, to determine if specific cutting andtranslocations happen. Un-guided assays have been developed allowingcomplementary testing of off-target cleavage including guide-seq. ThegRNA or pairs of gRNA with significant activity can then be followed upin cultured cells to measure correction of the HFE mutation. Off-targetevents can be followed again. Similarly hepatocytes can be transfectedand the level of gene correction and possible off-target eventsmeasured. These experiments allow optimization of nuclease and donordesign and delivery.

Example 99—Testing of Preferred Guides in Cells for Off-Target Activity

The gRNAs having the best on-target activity from the TIDE and nextgeneration sequencing studies in the above example will then be testedfor off-target activity using whole genome sequencing. Candidate gRNAswill be more completely evaluated in hepatocytes or iPSCs.

Example 100—Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, mutation correction and knock-in strategies will be tested forHDR gene editing.

For the mutation correction approach, donor DNA template will beprovided as a short single-stranded oligonucleotide, a shortdouble-stranded oligonucleotide (PAM sequence intact/PAM sequencemutated), a long single-stranded DNA molecule (PAM sequence intact/PAMsequence mutated) or a long double-stranded DNA molecule (PAM sequenceintact/PAM sequence mutated). In addition, the donor DNA template willbe delivered by AAV.

For the cDNA knock-in approach, a single-stranded or double-stranded DNAhaving homologous arms to the 6p21.3 region may include more than 40 ntof the first exon (the first coding exon) of the HFE gene, the completeCDS of the HFE gene and 3′UTR of the HFE gene, and at least 40 nt of thefollowing intron. The single-stranded or double-stranded DNA havinghomologous arms to the 6p21.3 region may include more than 80 nt of thefirst exon of the HFE gene, the complete CDS of the HFE gene and 3′UTRof the HFE gene, and at least 80 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to the6p21.3 region may include more than 100 nt of the first exon of the HFEgene, the complete CDS of the HFE gene and 3′UTR of the HFE gene, and atleast 100 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 6p21.3 region mayinclude more than 150 nt of the first exon of the HFE gene, the completeCDS of the HFE gene and 3′UTR of the HFE gene, and at least 150 nt ofthe following intron. The single-stranded or double-stranded DNA havinghomologous arms to the 6p21.3 region may include more than 300 nt of thefirst exon of the HFE gene, the complete CDS of the HFE gene and 3′UTRof the HFE gene, and at least 300 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to the6p21.3 region may include more than 400 nt of the first exon of the HFEgene, the complete CDS of the HFE gene and 3′UTR of the HFE gene, and atleast 400 nt of the following intron. Alternatively, the DNA templatewill be delivered by AAV.

Example 101—Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for HDR gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed in primary humanhepatocytes for efficiency of deletion, recombination, and off-targetspecificity. Cas9 mRNA or RNP will be formulated into lipidnanoparticles for delivery, sgRNAs will be formulated into nanoparticlesor delivered as AAV, and donor DNA will be formulated into nanoparticlesor delivered as AAV.

Example 102—In Vivo Testing in Relevant Animal Model

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, thelead formulations will be tested in vivo in a FRG mouse model with thelivers repopulated with human hepatocytes (normal or HFE deficient humanhepatocytes).

Culture in human cells allows direct testing on the human target and thebackground human genome, as described above.

Preclinical efficacy and safety evaluations can be observed throughengraftment of modified mouse or human hepatocytes in FRG mice. Themodified cells can be observed in the months after engraftment.

Example 103—In Vitro Transcribed (IVT) gRNA Screen

To identify a large spectrum of pairs of gRNAs able to edit the cognateDNA target region, an in vitro transcribed (IVT) gRNA screen wasconducted. The HFE genomic sequence, located on Chromsome 6 (6p21.3region), was submitted for analysis using a gRNA design software. Theresulting list of gRNAs were narrowed to a list of about 200 gRNAs basedon uniqueness of sequence (only gRNAs without a perfect match somewhereelse in the genome were screened) and minimal predicted off targets.This set of gRNAs were in vitro transcribed, and transfected usingmessenger Max into HEK293T cells that stably express Cas9. Cells wereharvested 48 hours post transfection, the genomic DNA was isolated, andcutting efficiency was evaluated using TIDE analysis. (FIGS. 2A-2D;FIGS. 3A-C).

Note Regarding Illustrative Examples

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various aspects of the presentinvention and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative aspects provided herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

What is claimed is:
 1. A method for editing the haemochromatosis (HFE)gene in a human cell by genome editing, the method comprising: (i)introducing into the human cell a Cas9 deoxyribonucleic acid (DNA)endonuclease or mRNA encoding the CAS9 DNA endonuclease, to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the HFE gene or within or near regulatory elements of theHFE gene that results in a permanent deletion, insertion or correctionof one or more mutations within or near the HFE gene and results in therestoration of HFE protein activity; and (ii) introducing into the cellone or more single-molecule guide ribonucleic acids (sgRNAs) comprisinga spacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 3610; 973; 378; 1029; 3598; 3634; 1142; 388;1121; 3678; 1137; 3531; 3654; 1045; 1096; 3558 and
 3585. 2. The methodof claim 1, wherein the method further comprises: introducing into thehuman cell a polynucleotide donor template comprising at least a portionof the wild-type HFE gene, at least a portion of DNA sequences thatencode wild-type regulatory elements of the HFE gene, or at least aportion of cDNA corresponding to the wild-type HFE gene.
 3. The methodof claim 1, wherein the method further comprises: introducing into thehuman cell a polynucleotide donor template comprising at least a portionof the wild-type HFE gene or regulatory elements of the HFE gene, andwherein the Cas9 DNA endonuclease effects one single-strand break (SSB)or double-strand break (DSB) at a locus within or near the HFE gene orwithin or near regulatory elements of the HFE gene, that facilitates theinsertion of the at least a portion of the wild-type HFE gene orregulatory elements of the HFE gene into chromosomal DNA that results ina permanent insertion or correction of the HFE gene or regulatoryelements of the HFE gene.
 4. The method of claim 1, wherein the methodfurther comprises: introducing into the human cell a polynucleotidedonor template comprising at least a portion of the wild-type HFE geneor regulatory elements of the HFE gene, wherein the Cas9 DNAendonuclease effects a pair of single-strand breaks (SSBs) ordouble-strand breaks (DSBs), the first at a 5′ locus and the second at a3′ locus, within or near the HFE gene or within or near the regulatoryelements of the HFE gene, that facilitates the insertion of the at leasta portion of the wild-type HFE gene or regulatory elements of the HFEgene into chromosomal DNA between the 5′ locus and the 3′ locus thatresults in a permanent insertion or correction of the HFE gene orregulatory elements of the HFE gene.
 5. The method of 3, wherein theCas9 DNA endonuclease is pre-complexed with the one or more sgRNAs. 6.The method of claim 1, wherein the method further comprises: introducinginto the human cell two of the one or more sgRNAs, and wherein the Cas9DNA endonuclease effects a pair of double-strand breaks (DSBs), thefirst at a 5′ DSB locus and the second at a 3′ DSB locus, within or nearthe HFE gene or within or near regulatory elements of the HFE gene, thatresults in a permanent deletion of the chromosomal DNA between the 5′DSB locus and the 3′ DSB locus, wherein the two sgRNAs are modifiedsgRNAs.
 7. The method of claim 1, wherein the Cas9 mRNA and the one ormore sgRNAs are either formulated into separate lipid nanoparticles orinto the same lipid nanoparticle, or wherein the Cas9 mRNA is formulatedinto a lipid nanoparticle and the one or more sgRNAs are introduced intothe human cell by an adeno-associated virus (AAV), or wherein the Cas9mRNA is formulated into a lipid nanoparticle and the one or more sgRNAsare introduced into the human cell by electroporation.
 8. Asingle-molecule guide ribonucleic acid (sgRNA) for editing thehaemochromatosis (HFE) gene in a cell from a patient with hereditaryhemochromatosis (HHC), comprising a spacer sequence selected from thegroup consisting of the nucleic acids sequences set forth by SEQ ID NOs:3610; 973; 378; 1029; 3598; 3634; 1142; 388; 1121; 3678; 1137; 3531;3654; 1045; 1096; 3558 and
 3585. 9. The method of claim 2, wherein theCas9 mRNA, the one or more sgRNAs, and the polynucleotide donor templateare either each formulated into separate lipid nanoparticles or into thesame lipid nanoparticle.
 10. The method of claim 2, wherein the Cas9mRNA is formulated into a lipid nanoparticle and both the one or moresgRNAs and the polynucleotide donor template are introduced into thehuman cell by an adeno-associated virus (AAV).
 11. The method of claim2, wherein the Cas9 mRNA is formulated into a lipid nanoparticle, theone or more sgRNAs are introduced into the human cell byelectroporation, and the polynucleotide donor template is introducedinto the human cell by an adeno-associated virus (AAV).
 12. The methodof claim 1, wherein the human cell is selected from the group consistingof a liver cell, skin cell, pancreatic cell, heart cell, joint cell, anda cell from the testes.
 13. The method of claim 1, wherein the Cas9 DNAendonucleases is pre-complexed with the one or more sgRNAs.
 14. Themethod of claim 2, wherein the at least a portion of the wild-type HFEgene or the at least a portion of cDNA corresponding to the wild-typeHFE gene is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7,intronic regions, or combinations thereof.
 15. The method of claim 2,wherein the polynucleotide donor template is either a single or doublestranded polynucleotide.
 16. The method of claim 6, wherein the Cas9 DNAendonuclease is pre-complexed with the two sgRNAs.